Compositions containing peroxometalate salts, in particular prophylactic and therapeutic pharmaceutical compositions

Abstract

The present invention relates to a mixture or composition having therapeutic activity, preferably for topical administration, comprising: at least one metal salt, wherein the metal is selected from the group consisting of molybdenum (Mo), tungsten (W), vanadium (V), gold (Au), lanthanides, in particular lanthanum; at least one chelating agent; at least one source of peroxy radicals; at least one buffer. And to a pharmaceutical composition constituted by the mixture or comprising the mixture, to a process for the preparation thereof and to the uses thereof, in particular in the method for the treatment of viral infections, in particular viral infections involving viruses of the Herpesviridae family, or in anti-inflammatory treatments.

Description

Technical Field

[0001] The present invention relates to active mixtures for therapeutic treatment, the mixtures comprising peroxométallate, such as peroxommolybdate and / or, for example, peroxománthide salts.

[0002] In particular, the present invention relates to active mixtures for the preventive and curative treatment of herpesvirus (Herpesviridae) infection, and especially to active mixtures for the preventive and curative treatment of infections with herpes simplex virus 1 (HSV-1 or HHV-1) and herpes simplex virus 2 (HSV-2 or HHV-2). The invention also relates to pharmaceutical compositions comprising such active mixtures, methods of preparation thereof, and uses thereof, particularly for therapeutic purposes. Background Technology

[0003] In infectious diseases, viral diseases, besides varying in severity (even fatal ones) depending on the type, also exhibit highly uneven incidence rates due to population and time, posing a major challenge to treatment. These pathogens require cellular hosts whose metabolites and components can be used for pathogen replication, making them difficult to eradicate through proactive treatment of healthy tissues relative to infected tissues, or even preventative treatment.

[0004] Among viral infections that affect humans, those caused by herpesviruses (Herpesviridae) are currently highly contagious and endemic. This DNA-containing viral family consists of eight viruses. Among them, HHV-1 or HSV-1 and HHV-2 or HSV-2 are the causes of oral herpes and vaginal-anal herpes.

[0005] All herpesviruses share clinical protein and genomic structural properties, such as latency, recurrent or lytic reactivation. As with all viral infections, treatment options are complex and often limited. This invention aims to address the technical problem of providing a pharmaceutical composition capable of combating viral infections, particularly those involving herpesviruses of the Herpesviridae family, and especially HSV-1 and HSV-2.

[0006] Infections caused by HSV-1 and HSV-2 are highly contagious (primarily through contact) and endemic (World Health Organization (WHO), January 2016, “The herpes virus,” archive number 400, http: / / www.who.int / mediacentre / factsheets / fs400 / fr / #). This database pertains to humans but not to any zoonotic diseases or any vectors. The prevalence of HSV-1 infection, which causes oral herpes, certain forms of genital herpes, and herpetic paronychia, varies depending on continent, diagnostic methods, and feasibility. It is estimated to be 84%–99% on average in Africa, 50%–100% in Asia, 65%–98% in Europe, and 57%–68% in North America. The prevalence of HSV-2 infection, which causes genital and anal herpes, varies depending on sex, social and ethnic characteristics, age, and continent. For example (see Centers for Disease Control and Prevention (CDC), 2010, MMW R Morb Mortal Wkly Rep., 59(15):456-459), it is estimated that in the United States, the seropositivity rate is 32% for women and 20% for men in the 40-49 age group.

[0007] Given their morbidity, HSV-1 and HSV-2 infections have a significant global impact. In fact, in terms of eradication, antiviral drugs, whether administered orally or parenterally, and regardless of whether they are pyrophosphate analogs (e.g., phosphonoformic acid), nucleoside analogs that are either non-activated or activated in situ (e.g., fecitabine, vidarabine, acyclovir and its prodrug valacyclovir), or helicase-primase complex inhibitors (prilatvir), are not completely effective curative treatments. They help reduce the severity and frequency of symptoms but do not eradicate the infection. Most often, due to the low solubility of these molecules, particularly ciclovir molecules (for acyclovir, 1.3 mg / mL in water at 25°C) and their limited intestinal absorption (approximately 20% orally for acyclovir), appropriate doses must be administered to patients.

[0008] Unlike topical ophthalmic medications, the clinical efficacy of anti-HSV substances used in dermatology (such as cilofovir, ibacitabin, and acyclovir) remains moderate and not constant.

[0009] In addition, acquired resistant forms of Herpes simplex virus have emerged that are resistant to conventional treatments via oral or parenteral routes. These are usually associated with mutations in the viral genome (Bacon TH et al., 2003, Clin. Microbiol. Rev., 16(1), 114-128; Biswas S. et al., 2008, Antiviral Res., 80(1), 81-85).

[0010] Although extremely rare, these infections can be complicated by severe and complex pathologies during viral reactivation, in addition to the patient's infectivity, pain, and recurring episodes, such as herpetic encephalitis or keratitis. Without treatment, the mortality rate from mother-to-child transmission can be as high as 60%. Herpes infection and its development can be associated with other pathologies, for example, in certain cancer cases or post-transplantation, due to the patient's immunosuppressive mechanisms (induced or pathological) or immunosuppression. HSV-2 is one of the most common infections (60%–90%) among people carrying HIV. The risk of contracting new HIV is three times higher in the presence of HSV-2 infection. Carriers of both infections have a higher risk of transmitting HIV. Summary of the Invention

[0011] Therefore, the object of the present invention is to solve the technical problem of providing a method for preparing such a pharmaceutical composition.

[0012] Another objective of this invention is to solve the technical problem of providing locally acceptable pharmaceutically active compositions.

[0013] Another objective of this invention is to solve the technical problem of providing pharmaceutically active compositions that are readily soluble in hydrophilic solvents.

[0014] A further objective of the present invention is to solve the technical problem of providing an active pharmaceutical composition during transepidermal transit.

[0015] A further objective of the present invention is to solve the technical problem of providing pharmaceutical compositions that are inactivated during transdermal access.

[0016] A further objective of the present invention is to solve the technical problem of providing pharmaceutical compositions whose biological phases can be well defined and obtained.

[0017] A further objective of this invention is to solve the technical problem of providing pharmaceutical compositions with the lowest bioavailability.

[0018] A further objective of this invention is to solve the technical problem of providing pharmaceutically active compositions that have anti-replication effects against viruses, particularly HSV.

[0019] A further objective of this invention is to solve the technical problem of providing pharmaceutically active compositions with high cytotoxicity to virus-infected cells, particularly HSV-infected cells.

[0020] A further objective of this invention is to solve the technical problem of providing pharmaceutically active compositions that have virtually no cytotoxicity to healthy cells.

[0021] A further objective of the present invention is to solve the technical problem of providing a pharmaceutically active composition having preventive efficacy, particularly against HSV.

[0022] A further objective of the present invention is to solve the technical problem of providing a pharmaceutically active composition that has preventive efficacy, particularly against HSV, especially during the prodromal phase of infection.

[0023] A further objective of the present invention is to solve the technical problem of providing a pharmaceutically active composition that has preventive efficacy, particularly by shortening the acute phase, and especially has preventive efficacy against HSV.

[0024] A further objective of the present invention is to solve the technical problem of providing pharmaceutically active compositions that can prevent or limit the infection process, and in particular, prevent or limit the HSV infection process.

[0025] A further objective of this invention is to solve the technical problem of providing pharmaceutically active compositions that exhibit good stability during their shelf life at room temperature and up to 45°C.

[0026] Description of the present invention

[0027] The inventors have discovered that the above-mentioned technical problems can be solved by providing a pharmaceutically active mixture or a therapeutically active mixture containing at least one peroxide metal salt.

[0028] "Pharmaceutical activity" refers to the fact that a mixture or composition has beneficial activity within the scope of therapeutic treatment.

[0029] The term "mixture" refers to a composition, but is not limited to compositions obtained by a particular preparation or manufacturing method. The term "mixture" as used herein provides the possibility of easily distinguishing "mixture" from "composition" from a semantic perspective, particularly when, for example, "mixture" is incorporated into a "composition" that contains other ingredients.

[0030] More specifically, according to a first aspect, the present invention relates to compositions or mixtures, preferably to compositions or mixtures having therapeutic activity via a local route, comprising:

[0031] At least one metal salt, wherein the metal is selected from molybdenum (Mo), tungsten (W), vanadium (V), gold (Au), and lanthanides, especially lanthanum;

[0032] At least one chelating agent;

[0033] At least one source of peroxy radicals;

[0034] At least one buffer.

[0035] Advantageously, the present invention relates to a composition or mixture, preferably to a composition or mixture having therapeutic activity via a local route, comprising:

[0036] At least one metal salt, wherein the metal is selected from molybdenum (Mo), tungsten (W), vanadium (V), gold (Au), lanthanides, especially lanthanum;

[0037] At least one chelating agent;

[0038] At least one source of peroxy radicals;

[0039] At least one buffer;

[0040] The mixture or composition preferably has a redox potential of 250 mV to 550 mV, more preferably 300 mV to 450 mV, and even more preferably 300 mV to 420 mV.

[0041] Advantageously, the mixtures according to the invention are capable of forming pharmaceuticals, especially stable and active pharmaceuticals in situ.

[0042] The mixtures according to the invention, in equilibrium with the aforementioned compounds, advantageously form peroxide metal complexes comprising their salts. The inventors, not wishing to be bound by theory, believe that the mixtures according to the invention can form stable, non-permanently equilibrium lattice-type supramolecular structures, thereby providing in-situ catalysts for Fenton and Haber-Weiss type reactions at the extracellular level. When the metals or other metals (not iron) in the mixtures of the invention undergo the aforementioned reactions, these reactions are referred to as "reaction-like reactions." Partially reactive substances temporarily generated by the active substances penetrate cells, more advantageously, infected cells.

[0043] According to one variant, the composition comprises several metal salts, wherein the metals are selected from molybdenum (Mo), tungsten (W), vanadium (V), gold (Au), lanthanides, and especially lanthanum;

[0044] Depending on the preparation method, the metal salt is a molybdenum salt or contains molybdenum.

[0045] Depending on the preparation method, the metal salt is a lanthanide salt or contains lanthanide elements.

[0046] Depending on the preparation method, the metal salt is a mixture of molybdenum salt and lanthanide salt.

[0047] Advantageously, the present invention relates to a composition or mixture, preferably a composition or mixture having therapeutic activity via a local route, comprising:

[0048] At least one molybdenum (Mo) salt, optionally combined with at least one salt of a metal selected from tungsten (W), vanadium (V), gold (Au), lanthanides, especially lanthanum;

[0049] At least one chelating agent;

[0050] At least one source of peroxy radicals;

[0051] At least one buffer;

[0052] The mixture or composition preferably has a redox potential of 250 mV to 550 mV, more preferably 300 mV to 450 mV, and even more preferably 300 mV to 420 mV.

[0053] Preferably, the metal salt exists in the mixture in the form of an oxide or a peroxide. Advantageously, the metal oxide in the mixture of the present invention can form a metal acid, more specifically an intermediate of a Lewis acid.

[0054] According to the alternative, metal compounds in which the oxidation state of the metal is compatible with metastable Fenton-Haber-Weis and Fenton-Haber-Weis "like" reactions (involving redox reactions of other metal ions that are not iron, in the case of chelation in this invention) are preferred, i.e., mainly transition metals, specifically Mo(VI), W(IV) to W(VI), V(III) to V(V), Au(I) to Au(III).

[0055] According to the alternative, preferred lanthanide compounds that can form LnO3-type sesquioxides are preferred, whose oxidation states are compatible with metastable Fenton-Haber-Wes and Fenton-Haber-Wes reactions, especially La(III), Ce(III) and Ce(IV), Nd(III) and Nd(IV), Sm(III) and Sm(IV), and Gd(IV).

[0056] Advantageously, the metal in the metal salt or lanthanide salt has the highest oxidation state.

[0057] Advantageously, the metal or lanthanide element is oxidized to its maximum oxidation degree.

[0058] According to a preferred alternative, the metal salt comprises molybdenum. Therefore, according to the alternative, the metal salt is a molybdenum salt. More specifically, the mixture according to the invention preferably comprises sodium molybdate. According to the invention, it is preferred to use a molybdenum salt with a valence state of VI. Advantageously, this salt can provide significant stability to the mixture of compositions. Furthermore, molybdenum having a reactive valence state (VI), compatible electronegativity, very low cytotoxicity, and the ability to undergo Fenton-Haber-Wesley reactions is particularly preferred, especially in the form of a salt, such as a sodium salt.

[0059] According to an alternative, the metal salt formed comprises a peroxymetalate, and more particularly in the form of a hydrated-peroxymetalate intermediate.

[0060] Advantageously, the molybdenum salt preferably has a peroxide unit or a hydrogenated peroxide unit. More advantageously, the active material is MoO4. 2- The form or its hydrogenated peroxy form (see Oyerinde Oyeyemi F. et al., Solution structure of molybdic acid from Raman spectroscopy and DFT analysis, Inorganica Chimica Acta 361 (2008) 1000–1007. Doi:10.1016 / j.ica.2007.06.025), for example, the intermediate complex of the Na4[Mo2O6(chelated)].10H2O type in the mixtures of the present invention.

[0061] According to an alternative, the metal and its salt are lanthanides or their salts.

[0062] According to alternatives, the formed metal salt comprises a peroxylanthanide acid salt, and more particularly in the form of a hydrogenated-peroxylanthanide acid salt intermediate. For example, it can also be a NaLn(chelate).xH2O2.yH2O (where x and y depend on the type of lanthanide (Ln) used in the mixture of the present invention) type intermediate complex.

[0063] Advantageously, molybdenum salts and lanthanide salts, and more particularly lanthanum salts, preferably have peroxy or hydrogenated peroxy units (see Subonitskiy, YL, Proshina, OP, Dyunin, AG et al., Thermodynamic properties of lanthanum Molybdates, Russ. J. Phys. Chem. (2016) 90: 267. Required: 10.1134 / S00360244160202031X).

[0064] Advantageously, the use of molybdenum salts can limit the cytotoxicity of the mixtures of the present invention, particularly to erythrocytes, lymphocytes, epithelial cells, fibroblasts and osteoblasts.

[0065] The advantage of using molybdenum is that it is a naturally occurring coenzyme factor (such as xanthine oxidase, glyceraldehyde-3-phosphate dehydrogenase, and ferroredoxin).

[0066] Advantageously, the use of lanthanum salts can limit the cytotoxicity of the mixtures according to the invention.

[0067] According to a preferred alternative, the metal in its reactive form is used to generate the reactive substance HO2. · And optional HO · and O2 · Catalyst.

[0068] According to an alternative, the mixture according to the invention contains 0.1 μM to 500 μM, preferably 1 μM to 200 μM, more preferably 5 μM to 150 μM of a metal salt.

[0069] According to an alternative, the mixture according to the invention contains 0.1 μM to 100 μM, preferably 1 μM to 50 μM, more preferably 5 μM to 30 μM of molybdenum metal salt.

[0070] According to an alternative, the mixture according to the invention contains 0.1 μM to 100 μM, preferably 1 μM to 50 μM, more preferably 5 μM to 30 μM of lanthanum metal salt.

[0071] The term "chelating agent (or chelate)" refers to a compound capable of forming a stable coordination complex with one or more ions, more specifically with a metal present in ionic form in a composition. The metal can be present in any form, including peroxide or hydrogenated peroxide forms. Such a complex is called a "chelate".

[0072] Advantageously, the chelating agent suitable for the mixtures of the present invention should be able to catalyze the in situ generation of active free radicals HO2 extracellularly. · (mainly HO2) · But there is also O2 · and OH · It can also chelate extracellular, bound or free calcium and metal ions, including ferrous ions (Fe(II) or Fe2+). 2+ ) and iron ions (Fe(III) or Fe 3+ Due to the effect of chelates on Ca... 2+ and Fe 2+The affinity of Fe for extracellular ions is significant, thus making the release of these reagents more efficient. These chelates are stable compounds in the pharmaceutically active mixtures of the present invention, and in addition to preferentially generating the free radical HO2, they also... · In addition, it also participates in the pharmaceutical or therapeutic activity of the mixtures of the present invention, particularly through preferential in-situ chelation of calcium and iron ions. This in-situ chelation can also regulate calcium influx induced on the one hand, especially by viral infection (e.g., herpesviruses), and on the other hand, by oxidative stress caused by the presence of free radicals or peroxides at the extracellular level.

[0073] Advantageously, the chelating agent does not penetrate the cell but remains outside the cell.

[0074] According to a preferred alternative, the chelating agent stabilizes the peroxy and peroxymolybdate complexes in the formulation. These peroxymolybdates and hydrogenated peroxymolybdates can be (Mo₂O₆). 4+ and [Mo4O] 12 (O2)2] 4+ type.

[0075] Advantageously, according to the alternative, the chelating agent can chelate the active substance ion MoO4. 2- (The sodium form of the active substance has a molecular weight of 205.937 g per mole) or peroxymolybdate (e.g., MoO3-BAPTAH2) 2- and / or MoO3-EGTAH2 2- Stabilize it (in the form of...).

[0076] According to an alternative, the mixture according to the invention contains at least two chelating agents.

[0077] Advantageously, the chelating agent binds by coordination with oxidized or peroxide metal salts in solution.

[0078] According to the alternative, when the metal salt is a molybdenum salt, the chelating agent complexes with an oxidized or peroxy metal acid salt to form an intermediate dimer complex [Mo₂O₆ (chelating agent)]. 4+ .10H2O and / or peroxytetramolybdate (VI) [Mo4O] 12 (O2)2] 4+ - Chelating agent.

[0079] According to the alternative, when the metal salt is a tungsten salt, the chelating agent complexes with an oxidized or peroxy metal salt to form a complex [W₂O₆ (chelating agent)]. 4+ .8H2O.

[0080] According to the alternative approach, when the lanthanide salt is a lanthanum salt, the chelating agent complexes with an oxidized or peroxy metal acid salt to form a complex [La(O2)-chelating agent].2+ .6H2O.

[0081] Advantageously, the chelating agent is compatible with the spatial capture of oxidized and / or peroxidized complexes of metal salts. More specifically, the size of the NN "bag" separating the carboxyl residues should be related to the coordination of the bag and the spatial folding of the carbon chain, and is advantageously compatible with the capture of intermediate metal complexes, especially in oxidized or peroxidized forms. Advantageously, the chelating agent is compatible with the charge and external atomic orbital structure of metal salts.

[0082] According to one aspect, the chelating agent is selected from organic polyacids (particularly amino-polycarboxylic acids) and their salts. Typically, amino-polycarboxylic acids comprise amine functional groups containing one or more nitrogen atoms, which are linked by atoms (usually carbon atoms and optionally oxygen atoms) to at least two carboxylic acid groups, preferably secondary or tertiary amines.

[0083] According to an alternative, the chelating agent has at least three coordinating groups, preferably carboxylic acid groups, at the three ends of the molecule forming the chelating agent, said coordinating groups being separated by a chain of at least three atoms (including at least one nitrogen atom).

[0084] Advantageously, the chelating agent has five coordinating groups, preferably carboxylic acid groups, at the five ends of the molecule forming the chelating agent, said coordinating groups being separated by a chain of at least six atoms, preferably nine atoms (including one nitrogen atom and preferably two nitrogen atoms separated by two atoms).

[0085] According to the alternative, the chelating agent has four coordinating groups, preferably carboxylic acid groups, at the four ends of the molecule forming the chelating agent, said coordinating groups being separated by a chain of at least six atoms, preferably twelve atoms (including two nitrogen atoms separated by at least eight atoms).

[0086] According to an alternative, the atoms separating the coordinating group and / or the nitrogen atom of the chelating agent are selected from carbon, nitrogen, and oxygen atoms. Some of the atoms separating the coordinating group and / or the atoms separating the nitrogen atom of the chelating agent may be included in one or more identical or different atomic rings, such as non-aromatic, aromatic, or heteroaromatic rings, for example, phenyl or pyridyl.

[0087] Advantageously, the chelating agent is selected from BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid), EGTA (ethylene glycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid), DTPA (diethylenetriaminepentaacetic acid), and any mixture thereof.

[0088] According to the alternative, the chelating agent is selected from BAPTA, EGTA and any mixture thereof.

[0089] Advantageously, BAPTA and / or EGTA can improve the stability of precursors and achieve better synthetic yields.

[0090] The pKa values ​​of the four carboxyl groups in BAPTA are: pK1 and pK2 < 4 (BAPTA is insoluble), pK3 = 5.47, and pK4 = 6.36; while the pKa values ​​of EGTA are: pK1 = 2.00, pK2 = 2.65, pK3 = 8.85, and pK4 = 9.46.

[0091] Advantageously, the pKa of the chelating agent is compatible with the pH of the solution of the present invention.

[0092] Advantageously, the pKa of the chelating agent is compatible with the pKa of the metal acid or metal peracid formed in the intermediate in the solution of the present invention.

[0093] Advantageously, the pKa of the chelating agent is compatible with the pH of the infected area (e.g., epithelium).

[0094] Advantageously, the selected chelating agents exhibit weak cytotoxicity. This is especially true for BAPTA and EGTA.

[0095] The amount and ratio of metal salt and chelating agent present are sufficient to produce a {peroxide-metal acid salt-chelating agent} complex.

[0096] Preferably, at least one of the chelating agents contains a greater affinity for calcium than for the metals used in the mixtures of the present invention. This allows for the advantageous replacement of the chelated complexes in the mixtures of the present invention with chelating agent-calcium complexes in vivo. Advantageously, the concentration of the chelate is related to the extracellular Ca2+ concentration. 2+ The concentration (2mM to 3mM in human plasma) is compatible, meaning that, from a therapeutic point of view, the amount of chelating agent contained in the mixture of the present invention is sufficient to effectively form a complex with calcium present in the extracellular space.

[0097] Advantageously, the chelating agent contained therein has a positive effect on iron (Fe) 2+ / Fe 3+ The affinity of Fe is greater than that for the metals used in the mixtures of this invention. This allows for the advantageous use of the chelating agent -Fe. 2+ / Fe 3+ The complex replaces the chelated complex of the mixture of the present invention within the complex body.

[0098] According to an alternative, the mixture according to the invention contains at least 0.0001 mM to 1 mM of a chelating agent.

[0099] According to an alternative, the mixture according to the invention contains 0.1 μM to 100 μM, preferably 1 μM to 50 μM, more preferably 5 μM to 20 μM of BAPTA. According to a variation, the mixture according to the invention contains 1 μM to 80 μM, more preferably 20 μM to 80 μM of BAPTA.

[0100] According to another alternative, the mixture of the present invention contains 0.1 mM to 1 mM, preferably 50 μM to 800 μM, more preferably 200 μM to 700 μM of EGTA. According to a variation, the mixture of the present invention contains 200 μM to 2000 μM, preferably 600 μM to 1400 μM of EGTA.

[0101] According to another alternative, the mixture of the present invention comprises BAPTA and EGTA at concentrations of 0.0001 mM to 1 mM, preferably 5 μM to 20 μM and 200 μM to 700 μM, respectively.

[0102] According to the alternative, the molybdenum salt and chelating agent are present in a ratio of 10 / 1 to 1 / 100 (ratio: Mo salt / chelating agent, expressed as molar concentration).

[0103] According to the alternative, the lanthanum salt and chelating agent are present in a ratio of 10 / 1 to 1 / 100 (ratio: La salt / chelating agent, expressed as molar concentration).

[0104] According to the alternative, molybdenum salt and BAPTA exist in a ratio of 3 / 1 to 1 / 10, expressed as molar concentration.

[0105] According to the alternative, molybdenum salt and EGTA exist in a ratio of 1 / 10 to 1 / 70, expressed as molar concentration.

[0106] According to the alternative, lanthanum salt and BAPTA exist in a ratio of 3 / 1 to 1 / 10, expressed as molar concentration.

[0107] According to the alternative, lanthanum salt and EGTA exist in a ratio of 1 / 10 to 1 / 70, expressed as molar concentration.

[0108] According to the alternative, the main free radical source is H2O2.

[0109] According to alternative solutions, one or more auxiliary radical sources or a small amount of in-situ synthesized radical sources may be added.

[0110] According to the alternative, H2O2 is an aqueous solution containing 110 volumes or 30% or 8.82 M H2O2.

[0111] Advantageously, the H2O2 source does not contain any stabilizers selected from: pyridine-carboxylic acids, such as pyridinecarboxylic acid; phosphonic acids and aminophosphonic acids; substituted amides or amines; alkyl sulfonic acids; and mixtures thereof.

[0112] According to the alternative, the main source of peroxide free radicals is hydrogen peroxide. Therefore, according to this alternative, the concentration range of hydrogen peroxide is 200 mM to 600 mM, preferably 300 mM to 500 mM, and more preferably 340 mM to 450 mM.

[0113] According to an alternative, the mixture according to the invention contains hydrogen peroxide in a proportion of 30 mM to 4.4 M, preferably 150 mM to 3 M, more preferably 235 mM to 1.8 M, and even more preferably 260 mM to 440 mM. According to an alternative, the mixture of the invention does not contain any temporarily added peracid.

[0114] Advantageously, the amount (or concentration) of hydrogen peroxide (H2O2) is determined by titration. Typically, this can be done using a potassium permanganate solution.

[0115] Hydrogen peroxide concentration can also be determined by UV spectroscopy.

[0116] Advantageously, a buffer is used to precisely control the pH of the mixture of the present invention.

[0117] Advantageously, a buffer is used to precisely control the pH during in-situ application of the solution according to the invention.

[0118] Carboxylic acid is preferred among buffers.

[0119] Preferably, the buffer does not contain the following acids: hydroxylated carboxylic acids (e.g., malic acid) or polycarboxylic acids (e.g., citric acid and isocitrate, malonic acid); carboxylic acids with a molecular weight greater than 100 g / mol, especially long-chain carboxylic acids (greater than C6), as they may cause cell membrane instability; and unsaturated aliphatic carboxylic acids (e.g., sorbic acid), as their alkenyl peroxidation can produce cytotoxicity.

[0120] According to an alternative, the present invention includes the presence of certain acids generated in situ during the synthesis process, preferably in small amounts (e.g., peracetic acid). These serve as auxiliary sources of free radicals in the manufacture of the present invention and for its therapeutic use.

[0121] According to alternatives, a buffer with a corresponding physiological (skin and mucous membrane) pH (i.e., pH values ​​of 4.4 to 5.0) and a pKa compatible with oxidizing and / or peroxy metal acids (in their ionized form) is preferred (e.g., MoO4H2, pk1 = 3.61 and pKa2 = 3.89).

[0122] Preferably, the buffer is present in the mixture of the present invention at a concentration of 1 mM to 500 mM, more preferably 10 mM to 200 mM, and even more preferably 50 mM to 100 mM.

[0123] The molar ratio of hydrogen peroxide to buffer is 2 to 9, preferably 3 to 8.

[0124] Advantageously, the mixture is buffered to obtain a pH of 4.4–5.0, which is compatible with: i) contact with skin and mucous membranes, ii) reactive substances such as MoO4. 2- Stability in the presence of H2O2, iii) Fenton-Haber-Weiss catalytic reactions or other catalytic reactions, and iv) in-situ generation of free radicals mainly of the HOO· type.

[0125] According to an alternative, the mixture contains a buffer, such as a carboxylic acid, and preferably acetic acid, to bring the pH to 4.0–5.2, preferably 4.4–5.0.

[0126] Advantageously, the mixture has a redox potential of 350 mV to 450 mV, preferably 350 mV to 420 mV.

[0127] The redox efficacy of the mixture of the present invention can also be improved by iron(III) or Fe. 3+ Quercetin is estimated by in vitro oxidation of ions. In particular, the determination method known as the "quercetin method" can be used (e.g., adapted from El Hajji H. et al. (2006) "Interactions of quercetin with iron and copper ions: Complexation and autoxidation", Free Radic. Res., 40(3), 303-320 and Balcerzak M. et al. (2008) "Selective determination of Fe(III) in Fe(II) samples by UV-spectrophotometry with the aid of quercetin and morin", Acta Pharm., 58, 327-334).

[0128] The present invention also relates to a method for preparing a mixture as defined in the present invention. More specifically, the present invention relates to a method for preparing a mixture of the present invention, comprising: (i) preparing a buffer solution (BS) containing a buffer having an acidic pH; (ii) preparing a solution (CS) containing a metal complex of a metal oxide salt; (iii) preparing a first initial solution (Si1) containing hydrogen peroxide; (iv) preparing a second initial solution (Si2) by mixing solution BS with solution Si1; (v) preparing a solution (S1) containing a peroxide metal compound by mixing solution CS with solution Si2; (vi) preparing solution S2 by adjusting the pH of solution S1 with an alkali, wherein the pH of solution S2 is more alkaline than the pH of solution BS; (vii) adding one or more chelating agents to solution S2; (viii) optionally, adjusting the pH; (ix) optionally, adjusting the volume of the final solution to obtain a pharmaceutically active mixture as defined in the present invention.

[0129] According to one variation, when using two or more metal oxide salts, solutions of metal complexes (SC) can be prepared using different metal oxides, including: preparing separate solutions of each metal oxide salt, and then mixing these solutions simultaneously or sequentially with a Si2 solution to prepare an S1 solution.

[0130] Preferably, the mixture according to the invention is prepared according to a catalytic synthesis method. The catalytic synthesis of the mixture according to the invention is generally sequential. Advantageously, the method of the invention comprises the sequence of steps (iii) => (iv) => (v) => (vi) => (vii) => (viii) => (ix).

[0131] Advantageously, the preparation method includes spectral analysis (e.g., by analyzing IR, UV and / or visible spectra) in one or more preparation steps, preferably in all preparation steps.

[0132] According to the alternative, the first initial solution Si1 has a first redox potential greater than that of the mixture according to the invention.

[0133] Preferably, after step (ix), the volume of the mixture is not diluted to avoid shifting the chemical equilibrium of the mixture according to the invention.

[0134] Of course, it is advantageous to use substances or compounds of sufficiently satisfactory purity as raw materials. In particular, different substances or compounds must be compatible with therapeutic uses.

[0135] According to alternative methods, the mixtures prepared according to the present invention can be subjected to IR-FT, NMR-H, and / or mass spectrometry analysis to ensure that the composition of the mixtures meets industrial requirements and is particularly suitable for pharmaceutical therapeutic use. It is also preferred that the precursor components of the mixtures of the present invention be subjected to IR-FT spectroscopic analysis.

[0136] According to alternative methods, pH can be controlled in one, several, or even all of the preparation steps.

[0137] According to the alternative, the redox potential can be controlled in one, several, or even all of the preparation steps.

[0138] According to alternative methods, the mixtures of the present invention can be controlled by density determination and / or UV spectrophotometry in one, several, or even all of the preparation steps.

[0139] According to the alternative approach, Fe2+ present or generated in vitro can be analyzed using the quercetin method (ultraviolet spectrophotometry). 3+ The redox potency of the mixture of the present invention was determined by in vitro oxidation of quercetin.

[0140] Advantageously, the concentration of hydrogen peroxide in solution Si1 is in the range of 200 mM to 600 mM, preferably 300 mM to 500 mM, and more preferably 330 mM to 460 mM.

[0141] According to the embodiments, the material used is metal, preferably stainless steel, and optionally passivated metal.

[0142] According to embodiments, the material can be repassivated during the preparation of the mixture of the present invention, for example by contacting the material (which will be in contact with the mixture or its precursor) with a hydrogen peroxide solution.

[0143] It is preferable to measure the hydrogen peroxide source to ensure the amount of hydrogen peroxide present and its role in preparing the mixture of the present invention. This measurement can be performed, for example, by titration or UV spectrophotometry (typically at 240 nm).

[0144] Advantageously, the pH of the buffer solution BS is 4.4 to 5.0, preferably 4.7 to 4.8.

[0145] Advantageously, the buffer in the BS solution is a carboxylic acid / carboxylate buffer, and the hydrogen peroxide / carboxylate ratio in the BS solution is 20 / 1 to 1 / 1, preferably about 5 / 1.

[0146] Advantageously, the ratio of acetic acid (AcOH) to the added acetate is limited to minimize the production of peracetic acid (AcOOH). Advantageously, in the preparation method according to the invention, the production of peracetic acid is minimized and its effect on catalysis is very small.

[0147] The preparation of the first initial solution Si1 involves preparing an aqueous solution of carboxylic acid, preferably an aqueous solution of acetic acid, followed by the addition of hydrogen peroxide.

[0148] Preferably, the first initial solution Si1 has a pH of 2.5 to 4.6, more preferably 2.7 to 4.2. Advantageously, the first initial solution Si1 has a redox potential of 400 mV to 550 mV, more preferably 420 mV to 500 mV.

[0149] Advantageously, the method includes measuring the redox potential in steps (iii) to (ix), and preferably also includes measuring the pH in steps (iii) to (ix).

[0150] According to an alternative, the second initial solution Si2 has a pH of 2.5 to 3.2, preferably 2.9. According to an alternative, the second initial solution Si2 has a redox potential greater than that of the first initial solution Si1. The redox potential of the second initial solution Si2 can be 420 mV to 570 mV, and is preferably 440 mV to 520 mV.

[0151] According to the alternative, the buffer solution BS is introduced into the first initial solution Si1.

[0152] According to one embodiment, the hydrogen peroxide concentration in the second initial solution Si2 is 300 mM to 500 mM, preferably 260 mM to 440 mM.

[0153] Advantageously, in step (iv), the pH is maintained below the permissible pH extreme for the catalytic synthesis reaction of the mixture according to the invention. Advantageously, a buffer is used in step (iv).

[0154] Advantageously, in step (iv), the peracetic acid produced in situ is minimal.

[0155] Advantageously, in step (iv), the redox potential of the Si1 solution in step (iii) is increased, for example by increasing the value from 440 mV to 460 mV to 460 mV in step (iv).

[0156] By monitoring the hydrogen peroxide concentration during the preparation of the mixture of the present invention, the generation of peroxide is observed during its introduction process, i.e., contacting the hydrogen peroxide with the buffer solution (step (iv)). Advantageously, hydrogen peroxide is added to an aqueous solution pre-buffered to an acidic pH. Thus, the first initial solution Si1 is prepared by adding hydrogen peroxide to a solution having an acidic pH, for example, pH 2.7 to 4.2.

[0157] Advantageously, step (iv) shifts the equilibrium toward the in-situ generation of H2O2, while simultaneously destroying the peracetic acid that may be produced by the chemical equilibrium.

[0158] According to the alternative, buffer solution BS is introduced into solution S1.

[0159] According to the alternative, the pH of the solution S1 containing the peroxide metal complex is 2.5 to 3.5, preferably 2.8 to 3.0.

[0160] According to one embodiment, the redox potential of the S1 solution containing the peroxide metal complex is 400mV to 550mV, preferably 440mV to 500mV.

[0161] According to one embodiment, the hydrogen peroxide concentration in the solution S1 containing the peroxide metal complex is 250 mM to 500 mM, preferably 320 mM to 450 mM.

[0162] In step (v), the added molybdate is in the form of molybdate MoO3·H2O and MoO3·2H2O.

[0163] Under conditions not specified in this invention, at room temperature, without any catalyst and without the addition of any strong acid (e.g., H₂SO₄), the active material MoO₄… 2 Synthesis under equilibrium conditions will take several days.

[0164] According to one embodiment, the pH of the S2 solution is buffered to a preferred value of 4.4 to 5.0, for example, 4.5 to 5.0. Advantageously, at this buffered pH, the unstable form of MoO4 is predominantly present. 2- (Mo's oxidation state (VI)), while hydrogen peroxide is stable. Hydrogen peroxide is typically stabilized industrially using phosphate buffers. Unstable phosphomolybdate intermediates (yellow) can form, which disappear at equilibrium, making H₂O₂ more reactive.

[0165] According to the alternative, the redox potential of the S2 solution is 300mV to 450mV, preferably 360mV to 410mV.

[0166] In the aqueous solution and at the pH used to synthesize the mixtures of the present invention, molybdenum (Mo(VI)) oxides can exist in the following different molecular structures: MoO4 2- and [MoO6] 2- [Mo2O3(O2)4(H2O)2] 2-However, it also exists in reactive polynuclear forms (e.g., the binuclear complex structure of oxygen-Mo(VI), (Dement'ev IA et al. (2007) "Mononuclear, polynuclear, and cluster complexes of molybdenum and their reactions as models of biochemical systems and processes", Russ.J.Gen.Chem.,77(5),822-843)).

[0167] Advantageously, when the metal salt used is a molybdenum salt, the preparation conditions allow for the in-situ formation of peroxymolybdate and hydrogenated peroxymolybdate. The latter is [Mo₂O₃(O₂)₄(H₂O)₂]. 2- According to an alternative, preferably, the molar amount of hydrogen peroxide is more than 1,000 times, more particularly more than 10,000 times, and more preferably 15,000 times, in excess of the metal salt, especially Mo(VI).

[0168] Advantageously, the chelating agent and the pH of the synthesis limit the movement of "Jones reducer" type reactions, which reduce metal salt ions (preferably molybdates) to a lower oxidation state of the metal (corresponding to metallic molybdenum). Alternatively, this movement is limited to weakly acidic aqueous media, typically at pH 4.4–5.0, for example 4.5–4.7.

[0169] Preferably, when several chelating agents are available, separate chelating agent solutions are prepared, each solution containing a specific chelating agent.

[0170] According to the preferred alternative, chelating agents are added in order of increasing pKa.

[0171] For example, it is possible to prepare a solution containing a BAPTA-type chelating agent on one hand, and a solution containing an EGTA-type chelating agent on the other hand. Preferably, BAPTA is added first, followed by EGTA.

[0172] Advantageously, the chelating agent can undergo, in particular, the Fenton-Haber-Weis reaction after local administration, with a specific dissociation constant Kd. 螯合 Fe 2+ / Fe 3+ >Ca 2+ .

[0173] Advantageously, chelating agents can reduce the main active free radicals HOO generated. · And a small amount of O2 · and HO · Extracellular in situ self-maintenance.

[0174] According to an alternative approach, BAPTA can first be added to solution S2 to obtain the resulting solution S3. For example, the pH range of solution S3 is 4.0–5.0, preferably 4.3–4.8. For example, the redox potential of solution S3 is substantially the same as that of solution S2. In other words, the redox potential does not change upon addition of a BAPTA-type chelating agent. According to an alternative approach, the redox potential of solution S3 is 250 mV–450 mV, preferably 300 mV–400 mV. Advantageously, BAPTA is pre-dissolved in a small amount of solution S2 before contacting it with the remainder of solution S2.

[0175] Preferably, the molar ratio of BAPTA concentration to molybdenum salt is 1 / 1 to 1 / 3, more preferably 1 / 2.

[0176] For example, the hydrogen peroxide concentration of the S3 solution is 250 mM to 500 mM, preferably 320 mM to 440 mM.

[0177] Advantageously, at least one other chelating agent of the EGTA type can be added to solution S3 to obtain the resulting solution S4. Alternatively, the pH range of solution S4 is 4.0–5.0, preferably 4.3–4.7. Alternatively, the redox potential of the solution is 300 mV–400 mV, preferably 350 mV–390 mV.

[0178] Preferably, the molar ratio of EGTA concentration to molybdenum salt is 10 / 1 to 50 / 1, more preferably about 25 / 1.

[0179] For example, the hydrogen peroxide concentration of the S4 solution is 250 mM to 550 mM, preferably 320 mM to 470 mM.

[0180] According to an alternative, the pH in step (viii) is adjusted using an alkaline solution, such as an alkaline solution of the sodium hydroxide type, preferably concentrated to, for example, an 8M to 15M concentration. Advantageously, the pH in step (viii) can be adjusted to 4.4 to 5.0, preferably to about 4.6.

[0181] According to an alternative, the pH of the mixture of the present invention is 4.50 to 4.70. Advantageously, the redox potential of the mixture of the present invention is 320 mV to 390 mV.

[0182] According to a preferred alternative, the method includes reducing the redox potential in step (vi). For example, the reduction in redox potential can be 50 mV to 150 mV, and is typically about 90 mV.

[0183] Preferably, after the redox potential decreases in step (vi), the redox potential is kept substantially constant.

[0184] Advantageously, the mixture according to the invention is stable over time for six months during storage, for example at room temperature or at temperatures up to 45°C. It is recommended to keep the mixture in the dark and away from moisture.

[0185] pH was measured using a calibrated pH meter with temperature compensation and a KCl 4M / AgCl combined electrode.

[0186] Redox potential is measured using a combination pH meter / redox meter equipped with a calibrated platinum / calomel electrode. Typically, this measurement is performed at room temperature (i.e., 20°C) and ambient pressure (i.e., 101325 Pa).

[0187] Preferably, the concentration of hydrogen peroxide in the above solution is estimated by UV spectroscopy according to an equation derived from Beer-Lambert's law: A 240nm = 43.6 × I × [H2O2], where I is in centimeters and [H2O2] is the molar concentration (Noble RW et al. (1970) "The reaction of ferrous horseradish peroxidase with hydrogenperoxide", J. Biol. Chem., 245(9), 2409-2413).

[0188] According to a specific alternative, the peroxide metal compound in solution S1 is a molybdenum peroxide compound.

[0189] Therefore, according to this alternative, the mixture of the present invention comprises a metal peroxide compound. According to a preferred alternative, the mixture of the present invention comprises a molybdenum peroxide complex.

[0190] According to an alternative, the solution contains sodium molybdate in the form of Mo(VI). Advantageously, at the pH of the solution of the present invention, the salt is primarily in the catalytic form MoO3-BAPTA. 3- and mainly MoO3-EGTA 3- Both are active substances, MoO4. 2- The source material.

[0191] According to specific embodiments, the mixture according to the invention comprises: a salt, preferably a sodium salt, a molybdenum salt; a mixture of two chelating agents, preferably of the BAPTA and EGTA type; hydrogen peroxide; and an acetate buffer (e.g., acetic acid / sodium acetate).

[0192] According to a specific alternative, the mixture according to the present invention comprises:

[0193] Molybdates, such as sodium molybdate;

[0194] BAPTA;

[0195] EGTA;

[0196] H2O2;

[0197] CH3COOH / CH3COONa;

[0198] E 氧化还原 The voltage range is 250mV to 550mV, preferably 300mV to 450mV, and more preferably 300mV to 420mV;

[0199] The pH ranges from 4.4 to 5.0.

[0200] According to a specific variant, the mixture according to the invention comprises:

[0201] Molybdates, such as sodium molybdate;

[0202] Lanthanum salts, such as lanthanum nitrate;

[0203] BAPTA;

[0204] EGTA;

[0205] H2O2;

[0206] CH3COOH / CH3COONa;

[0207] E 氧化还原 The voltage range is 250mV to 550mV, preferably 300mV to 450mV, and more preferably 300mV to 420mV;

[0208] The pH ranges from 4.4 to 5.0.

[0209] Advantageously, the compounds in the mixture have the required amounts, pH conditions, and redox potentials to form a peroxymolybdate complex or a hydrogenated-peroxymolybdate complex in which the oxidation state of molybdenum is VI.

[0210] According to specific embodiments, the mixture of the present invention contains at least one chelating agent, preferably at least two chelating agents, and preferably their concentrations are 5 μM to 20 μM and 200 μM to 700 μM, respectively.

[0211] According to specific embodiments, the mixture of the present invention contains 340mM to 450mM of a major free radical donor, such as H2O2.

[0212] This invention particularly relates to compositions according to the invention as pharmaceutical compositions, said compositions comprising at least one metal salt selected from molybdenum (Mo), tungsten (W), vanadium (V), gold (Au), lanthanides, especially lanthanum; at least one chelating agent; and a peroxide radical source. The compositions according to the invention can be used in therapeutic treatments.

[0213] The present invention also relates to a method of therapeutic treatment, comprising advantageously administering a therapeutically effective amount of the pharmaceutical composition of the present invention to a subject in need by topical application.

[0214] The present invention also relates to a method for preparing a pharmaceutical composition according to the invention for use in therapeutic treatment methods.

[0215] The present invention particularly relates to pharmaceutical compositions comprising or composed of mixtures according to the invention.

[0216] Therefore, the present invention relates to pharmaceutical compositions for topical use, characterized in that the pharmaceutical composition comprises a therapeutically active mixture as defined in the present invention or a therapeutically active mixture obtainable by a method as defined in the present invention.

[0217] Advantageously, the pharmaceutical composition according to the invention comprises 0.001 mM to 5 mM, preferably 0.01 mM to 2 mM, more preferably 0.02 mM to 1 mM of a pharmaceutically active mixture.

[0218] This invention particularly relates to topical pharmaceutical compositions according to the invention for the treatment of viral infections, especially for the treatment of viruses of the Herpesviridae family.

[0219] The present invention particularly relates to a topical pharmaceutical composition according to the invention for a therapeutic treatment of infections involving HSV-1 and / or HSV-2.

[0220] This invention particularly relates to a topical pharmaceutical composition according to the invention for a therapeutic method of treating inflammation.

[0221] This invention particularly relates to topical pharmaceutical compositions according to the invention for use as a preventative or curative treatment method.

[0222] The present invention also relates to pharmaceutical compositions comprising molybdenum, at least one chelating agent, and a peroxide free radical source for therapeutic treatment of herpes. This composition is defined individually or in any combination by any embodiment, alternative, advantageous, preferred, or example of the invention.

[0223] The term "according to the invention" means any embodiment, alternative, advantageous mode, preferred mode or example of the invention, whether referred to individually or in combination.

[0224] Typically, the topical pharmaceutical compositions according to the present invention contain excipients, particularly pharmacopoeia-approved excipients.

[0225] Anti-infective therapeutic treatment refers to preventive, curative, and preventative treatments that can limit infection, such as in cases of contact infection, disease onset, or acute phase of disease, and that may partially or not eradicate the causative pathogen and / or limit its spread.

[0226] Advantageously, the active mixtures or pharmaceutical compositions according to the invention have in-situ activity during their use.

[0227] Advantageously, the active mixture or pharmaceutical composition according to the invention is active during transepithelial passage, and preferably transepithelial passage is not permitted or is limited.

[0228] Advantageously, the active mixture or pharmaceutical composition according to the invention is inactivated during transdermal access.

[0229] According to an alternative, the mixture or composition comprising the invention is applied topically to the oral and facial regions.

[0230] According to an alternative, the mixture or composition comprising the invention is applied topically to the genitals or anus.

[0231] According to an alternative, the mixture or composition comprising the invention is applied to the skin via a topical route.

[0232] According to an alternative, the mixture or composition comprising the invention is applied topically to the mucous membrane.

[0233] According to the alternative, the European Pharmacopoeia (Ph.Eur.), national pharmacopoeias (especially USP, JP, IP, Ph.Helv, Ph.Belg, Ph.Fr., BP, DAB, etc.) shall be used. Excipients approved by the International Pharmacopoeia (Ph.Int.;WHO) may be introduced during the preparation of this invention and / or introduced into the “mixture” designated as “mixture 1”.

[0234] The mixtures and compositions of the present invention have pharmaceutical activities particularly suitable for humans.

[0235] Advantageously, the active mixtures of the present invention are capable of undergoing Fenton-Haber-Wesley and Fenton-Haber-Wesley-like reactions, and preferentially generate free radicals HO2. · and a small number of free radicals O2· HO · .

[0236] According to the inventor, the reactive substance HOO · Its penetration is sufficient to induce apoptosis in a limited and controlled manner.

[0237] According to the inventor, the reactive substance HOO · Its penetration is sufficient to have an effective and controlled anti-replication effect on the virus.

[0238] According to the present invention, the redox potential of the mixture is preferably used to limit the concentration of the free radical active substance precursor within a narrow range, thereby achieving a good balance between: anti-replication effect / moderate cytotoxicity to healthy cells / significant cytotoxicity to infected cells / infection prevention effect.

[0239] Advantageously, the therapeutic or pharmaceutically active mixture according to the invention enables the primary and preferably secondary generation of reactive free radicals HOO. · .

[0240] Therefore, according to the present invention, extracellular calcium is produced in vivo from HOO. · The main initiating factor. For example, when the mixture of the present invention contains molybdenum salt and BAPTA, it is mainly the complex 2Mo. (VI) -BAPTA2(H2O2) plays a role. For example, when the mixtures of the present invention contain molybdenum salt and EGTA, the main complex 2Mo is the primary component. (VI) -EGTA2(H2O2) plays a role.

[0241] Advantageously, HOO generated in the extracellular medium · This enables extracellular modifications (e.g., oxidation of extracellular amino acid residues), thereby limiting viral infection by modifying cellular recognition.

[0242] Advantageously, HOO generated in the extracellular medium · and its cell permeability and relative to OH · or O2 · It exhibits a long lifetime (≥1s) and good cell penetration compatibility (1ms penetration and approximately 1nm penetration). Advantageously, this substance HOO · It participates in the Hubble-Wesley response and Fenton response ("class-" or not" class-") at both extracellular and intracellular levels and allows the latter to be self-sustaining.

[0243] Advantageously, the Mo(IV)-containing mixture according to the invention has a pH of 4.4 to 5.0. This is particularly effective for HO2 · / O2 · The rate constant k of the decompositionobs (M -1 .s -1 Optimization.

[0244] Advantageously, altering the intracellular redox potential through contact with the composition according to the invention enables an apoptosis / anti-apoptosis balance. More specifically, this apoptosis / anti-apoptosis balance depends particularly on the presence of HSV infection, primarily caused by HSV-1 and HSV-2.

[0245] According to the alternative, the mixture of the present invention is selectively active against cells infected with HSV, primarily HSV-1 and HSV-2.

[0246] Therefore, there is a close balance between the regulation of stress pathways induced by HSV infection (including calcium influx) and apoptosis.

[0247] The presence of intracytoplasmic free radicals and changes in cellular redox potential influences this balance. This induces the same cellular stress pathways, the same calcium influx, and the same induced metabolic pathways as in HSV infection, which are then reverse-regulated to a different anti-apoptotic balance. This supports the therapeutic effects of the mixtures or compositions comprising the present invention.

[0248] According to an alternative, the pharmaceutical composition or mixture of the present invention is used in a therapeutic method for treating cold sores and oral herpes.

[0249] According to an alternative, the pharmaceutical composition or mixture according to the invention is used in a therapeutic method for treating genital and anal herpes.

[0250] According to embodiments, the active mixtures according to the invention possess anti-infective potency (as potential candidates, moderate to low direct action against viruses and viral receptors in cells), anti-recognition, and anti-internalization (the important role of cellular receptors in viruses). These potency occur outside the cell and limit viral infectivity. Therefore, the active mixtures according to the invention have a preventative effect. Similarly, the active mixtures according to the invention possess anti-infective potency.

[0251] According to an alternative, the pharmaceutical composition or mixture according to the invention is used in the therapeutic treatment of populations expressing, replicating, or infectious HSV.

[0252] According to an alternative, the pharmaceutical composition or mixture according to the invention is used in the therapeutic treatment of cell populations or tissues expressing HSV-1 (Kessler HH et al. (2000) "Detection of Herpes Simplex Virus DNA by real-time PCR", J Clin Microbiol., 38(7), 2638-2642).

[0253] According to an alternative, the pharmaceutical composition or mixture according to the invention is used in the therapeutic treatment of cell populations or tissues expressing HSV-2 (Kessler HH et al. (2000) "Detection of Herpes Simplex Virus DNA by real-time PCR", J Clin Microbiol., 38(7), 2638-2642).

[0254] Advantageously, the active mixture according to the invention has anti-replication potency induced by prior contact between healthy cells or tissues and the infection. The active mixture according to the invention can be used for infection prevention by limiting intracellular viral replication and / or by limiting viral infiltration into cells.

[0255] Advantageously, the active mixture according to the invention has preferential anti-replication efficacy against cellular metabolism (oxidative stress pathways and calcium channels) of infected cells. The active mixture according to the invention can be particularly effective against intracellular viral replication directly and against HSV-1 and / or HSV-2 overinfection of cells in infected tissues and adjacent healthy tissues.

[0256] According to an alternative, the active mixture according to the invention non-uniformly induces the inhibition of interleukin-6 (IL-6) production in human skin flaps, thus exhibiting anti-inflammatory efficacy. Attached Figure Description

[0257] In the diagram:

[0258] Figure 1 The changes in redox potential and H2O2 concentration during the preparation of mixture 1 are shown.

[0259] Figure 2 Fe is shown as used to determine the redox efficiency of mixture 1. 3+ Absorption diagram (abaque).

[0260] Figure 3 The redox potency of mixture 1 at different concentrations was shown (in human plasma culture medium compared with that of mixture 1 (in nM MoO4)). 2-(This indicates the absorption diagram of Fe(III) in quercetin after contact (2 minutes).)

[0261] Figure 4 Absorption diagrams of the redox efficacy of mixture 1 at different concentrations are shown.

[0262] Figure 5 Different MoO4 were shown 2- The following effects of the concentration of mixture 1: i) after contact with the formulation, on the level of transferrin-Fe in human plasma. 3+ In-situ generation of Fe from complex 3+ The effect, ii) on Fe 2+ / Fe 3+ The effects of balance and iii) on the oxidation of quercetin.

[0263] Figure 6 Interleukin-6 (IL-6) produced in the culture supernatant of a nonpathological human skin biopsy (2 min) stimulated with mixture 1 (24.3 μM of active substance) or NaCl (100 mM) as a control. (D1-5: donor 1 to donor 5, B1 or 2: biopsy 1 or 2).

[0264] Figure 7 The average interleukin-6 (IL-6) production of culture supernatant from non-pathological human skin biopsies (2 min) stimulated with mixture 1 (24.3 mM active substance) or NaCl (100 mM) as a control is shown at 6 h and 24 h after exposure.

[0265] Figure 8 The redox efficiencies of mixtures 1 and 2 at different concentrations are shown (in comparison with mixture 1 (nMMoO4)). 2- (middle) or mixture 2(nM La(MoO4) 1- (Graph showing the Fe(III) uptake of quercetin in human plasma culture medium after contact (2 minutes)). Detailed Implementation

[0266] Example

[0267] Example 1 - Example of an active mixture according to the present invention

[0268] This example was prepared from a molybdenum salt. The composition (“Mixture 1”) was prepared according to the proportions in Table 1, which shows the initial concentrations of each component:

[0269] Table 1

[0270] Element The mixture according to the invention Sodium molybdate 20.7μM BAPTA 12.6μM EGTA 526μM <![CDATA[H2O2]]> 353mM <![CDATA[CH3COOH / CH3COONa]]> 70mM pH (adjusted by NaOH) 4.4～5.0 <![CDATA[E° 氧化还原 ]]> 300mV~420mV

[0271] This composition is referred to as the “initial” composition because it corresponds to the concentration of the added reagent, without taking into account the catalytic process that occurs.

[0272] All components used in the synthesis and the final product were verified by IR-FT spectroscopy.

[0273] Table 1a

[0274] product purity Molecular formula CAS number Sodium molybdate dihydrate 98％～103％ <![CDATA[Na2MoO4·2H2O]]> 10102-40-6 BAPTA: (1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) ≥98％ <![CDATA[C 22 H 24 N2O 10 ]]> 85233-19-8 EGTA: (ethylene glycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid) ≥99％ <![CDATA[C 14 H 24 N2O 10 ]]> 67-42-5 hydrogen peroxide 29％～31％ <![CDATA[H2O2]]> 722-84-1 glacial acetic acid 99.8％～100.5％ <![CDATA[CH3COOH]]> 64-19-7 Sodium acetate trihydrate 99％～101％ <![CDATA[C2H3NaO2·3H2O]]> 5010524 Sodium hydroxide 10N NaOH 1310-73-2 softened water 1μS <![CDATA[H2O]]> 7732-18-5

[0275] Data measured during and after synthesis are averages from three products.

[0276] The quantities given below are for preparing 1 liter of mixture 1.

[0277] A Step (i): Prepare the buffer solution (Solution BS)

[0278]

[0279] Room temperature (20℃)

[0280] B- Step (ii): Preparation of the metal complex solution (solution CS)

[0281] Na₂MoO₄·2H₂O (Mo(VI) source; 100%) 200mg (final concentration 2.4mM)

[0282] Water qsp 340ml

[0283] Stir gently at room temperature

[0284] C -Step (iii): Preparation of the initial solution (Si1)

[0285] 900ml of water

[0286] CH3COOH (100%) 4mL (70mM)

[0287] Add very slowly, stirring very gently (250 rpm).

[0288] pH: 2.9–3.8

[0289] E 氧化还原 470mV~490mV

[0290] Then,

[0291] H2O2 (30%) qsp, the final concentration is 1.2wt% according to preliminary determination.

[0292] At 45 minutes:

[0293] pH: 2.70~2.90

[0294] E 氧化还原 440mV~460mV

[0295] According to the Nobel equation: [H₂O₂] = 380 mM ~ 410 mM (solution Si₁)

[0296] D Step (iv): Preparation of the initial solution (Si2)

[0297] Add 12 mL of "BS solution" to solution Si1 (≈1 mM acetate).

[0298] Add and stir gently.

[0299] At 45 minutes:

[0300] pH: ≈2.90

[0301] E 氧化还原 460mV~480mV

[0302] According to Nobel's equation: [H₂O₂] = 360mM~410mM (Si₂ solution)

[0303] E- Step (v): Preparation of peroxymolybdic acid solution S1

[0304] 10 mL of the "CS solution" was introduced into the Si2 solution.

[0305] At 45 minutes:

[0306] pH: 2.80~3.00

[0307] E 氧化还原 450mV~480mV

[0308] Using Nobel's equation: [H₂O₂] = 370 mM ~ 400 mM (solution S1)

[0309] F- Step (vi): Prepare solution S2

[0310] Adjust the pH value with gentle stirring:

[0311] For a 0.9 L mixture 1, use 3.6 mL of NaOH (9.9 M to 10.1 M).

[0312] pH: 4.5–5.0

[0313] At 45 minutes:

[0314] pH: 4.60

[0315] E 氧化还原380mV~390mV (solution S2)

[0316] G- Step (vii): Add chelating agent - prepare solution S4

[0317] Introducing BAPTA (98.8%) 6 mg / L

[0318] BAPTA was dissolved very slowly with gentle stirring in a small amount of formulation S2 (1.4 L) that had been pre-equilibrated at 25°C (referred to as "S3.1" solution).

[0319] Then, pour solution S3.1 into solution S2 to form solution S3.2.

[0320] At 45 minutes:

[0321] pH: 4.40–4.70

[0322] E 氧化还原 380mV~390mV

[0323] Using Nobel's equation: [H₂O₂] = 370mM~390mM

[0324] 200 mg / L EGTA (99.1%) was introduced into solution S3.2 to form solution S4.

[0325] Dissolve with gentle stirring.

[0326] At 45 minutes:

[0327] pH: 4.40–4.60

[0328] E 氧化还原 380mV~390mV

[0329] Using Nobel's equation: [H₂O₂] = 370mM~420mM

[0330] H- Step (viii): Adjust the pH value

[0331] Adjust pH (NaOH 9.9M~10.1M, i.e. ≈400g / L) => pH: 4.60±0.2

[0332] I- Step (ix): Adjust the volume of the final solution (FS)

[0333] Adjust the volume using softened water (qsp 1 liter).

[0334] During the 12-18 hour period:

[0335] pH: 4.50–4.70

[0336] E氧化还原 380mV~390mV

[0337] Using Nobel's equation: [H₂O₂] = 350mM~380mM

[0338] By titration: [H₂O₂] = 400mM~430mM

[0339] Density: δ = 1.004 g / ml

[0340] The ready-to-use mixture according to the invention is stable for more than 6 months in the dark at room temperature or 45°C.

[0341] In the final mixture 1, the concentration of hydrogenated peroxymolybdate sodium was 24.3 μM.

[0342] Example 2 - Characterization by changing redox potential

[0343] During the synthesis of the composition prepared according to Example 1, the characteristics of the active mixture according to the invention were evaluated by changing the redox potential.

[0344] according to Figure 1 The redox potential decreased by approximately 90 mV (pH adjusted with baking soda) in step 6, relative to the initial solution (≈470 mV; step 3). The redox potential was kept constant until the end of the synthesis (step 9). The formulation of the mixture of the present invention according to Example 1 was aged for 6 months at room temperature (389 mV ± 5 mV) and 45°C (389 mV ± 8 mV), respectively, at pH values ​​of 4.56 ± 0.40 and 4.53 ± 0.41.

[0345] Example 3 - Characterization by consumption and generation of H2O2 - Peroxide generated in mixture 1 - Reagent equilibrium and redox potential.

[0346] During the synthesis of the composition prepared according to Example 1, the characteristics of the active mixture according to the invention were evaluated by the consumption and generation of H2O2.

[0347] according to Figure 1 Relative to the initial H2O2 concentration (353 mM; steps 3–10), rapid formation of peroxides (≈42 mM; including a small portion of peracetic acid) was observed due to the addition of H2O2 to the buffer solution (acetic acid / acetate). If H2O2 is not added to the pre-acid-buffered aqueous solution (step 3; pH = 2.84 ± 0.1), the H2O2 will be generated due to the excessively high pH (water) (pKa). HO2·- / O2·- :4.8) and degrade spontaneously and rapidly.

[0348] From step 4 to step 7, limited consumption of H2O2 (10 mM ± 6 mM) was observed until it stabilized at 381 mM ± 5 mM (step 7; +BAPTA).

[0349] The addition of EGTA disrupted the peroxide equilibrium (Fenton-like reaction) through peroxide degradation (i.e., 393mM±29mM in step 7; 357mM±26mM in step 9; and 365mM±15mM after step 9+12 hours).

[0350] In step 6, a sharp decrease in redox potential was observed by adjusting the pH (from 463mV ± 15mV to 392mV ± 17mV). The sequential addition of the chelating agents BAPTA and EGTA (step 7) helped stabilize the redox potential (386mV ± 5mV and 383mV ± 6mV) by stabilizing the chemical equilibrium.

[0351] Example 4 - Determination of the redox potency of the mixture of the present invention: quercetin method.

[0352] A- reaction solution

[0353] 1- Quercetin

[0354] Preparation of quercetin dihydrate (2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-1-benzopyran-4-one dihydrate, 3,3',4',5,7-pentahydroxyflavone dihydrate (C 15 H 10 O7·2H2O) with 10 -3 A solution of mol / L in methanol.

[0355] 2- hydrochloric acid solution

[0356] A 1M hydrochloric acid first solution was prepared in methanol. A 0.3M second solution was prepared in methanol. The reaction solution was prepared by adding 343 μl of the 1M solution to 1357 μl of the 0.3M solution.

[0357] 3- Fe 3+ solution (reference absorbance)

[0358] HPLC preparation of 2 mg / ml Fe in water 3+(Fe2(SO4)3·xH2O) solution. This solution was then diluted to obtain eight reference solutions of the following concentrations: 20 μg / ml, 100 μg / ml, 200 μg / ml, 300 μg / ml, 400 μg / ml, 500 μg / ml, 1000 μg / ml, and 1500 μg / ml. Finally, these solutions were diluted 20-fold in human plasma or HPLC-grade water to achieve the following final concentrations: 0 μg / ml, 1 μg / ml, 5 μg / ml, 10 μg / ml, 15 μg / ml, 20 μg / ml, 25 μg / ml, 50 μg / ml, and 75 μg / ml.

[0359] 4- Fe 2+ of the solution (reference absorbance, active control)

[0360] Fe 89 mg / ml was prepared in water by HPLC. 2+ (FeSO4·7H2O) solution. This solution was then diluted to obtain three reference solutions with the following concentrations: 0.445 mg / ml, 4.45 mg / ml, and 44.5 mg / ml. Finally, these solutions were diluted 10-fold in water for HPLC to achieve the following final concentrations: 0 mg / ml, 0.445 mg / ml, 0.445 mg / ml, and 4.45 mg / ml.

[0361] B- The determination was performed using the quercetin method.

[0362] Quantification of Fe using the quercetin method 3+ The presence (absorbance) or the generation (Fe) 2+ Absorbance and efficacy of the mixture of the present invention after 2 minutes of contact.

[0363] Based on the analytical conditions or the mixture used:

[0364] "mixture 氧化 "" refers to the mixture according to the present invention as an oxidizing agent.

[0365] "mixture 还原 "" refers to the mixture according to the present invention, which acts as a reducing agent.

[0366] Fe is generated as follows 3+ :

[0367] Transferrin-2Fe 3+ + Mixture 还原 +3H + →Transferrin 还原 +2Fe 3+ (aq) + mixture 氧化 (1)

[0368]

[0369] Fe 3+ (aq) + Quercetin 还原 →Quercetin 氧化 +Fe 2+ (aq) (3)

[0370] Reaction (1) was carried out in a plasma medium.

[0371] Reaction (2) is carried out in plasma medium or HPLC water and corresponds to the equilibrium of the Fenton-Haber-Weiss reaction.

[0372] Reaction (3) is the oxidation reaction of quercetin, used to evaluate the redox potential of mixture 1.

[0373] In an acidic medium and at 70°C, quercetin is converted into ferric iron (Fe3+) carried by transferrin in a plasma medium. 3+； ElHajji et al. (2006) and Balcerzak et al. (2008) specifically oxidize without being affected by ferrous iron (Fe²⁺). 2+ Oxidation. Under these conditions, Fe... 3+ The ions are soluble.

[0374] Add 30 μl of quercetin solution to 170 μl of hydrochloric acid reaction solution. After homogenization, add 50 μl of sample (Fe from human plasma or HPLC-reacted water). 3+ Fe in water by HPLC 2+ (The mixture of the present invention after contact with human plasma for 2 minutes). The solution was vigorously stirred briefly, then incubated at 70°C for 1 hour. After incubation, the sample was centrifuged at 14000 rpm for 15 minutes at room temperature. 100 μl of the supernatant was collected. The UV spectrum of each sample was obtained at 230 nm–500 nm. Blank water and Fe were removed according to the experimental type. 2 + Fe 3+ Plasma. Retains Fe produced by plasma transferrin. 3+ The absorption peaks of quercetin oxidized in situ by the in situ Fenton-Haber-Weis reaction (from 285 nm to 305 nm).

[0375] When experiments are performed in water or plasma using HPLC, the center of the curve peak is around 292 nm. When the experimental blank is removed, it can shift by up to 10 nm.

[0376] 1-Fe 3+ Absorption diagram

[0377] For example, by analyzing different concentrations of Fe in human blood plasma 3+ (See Fe above) 3+The UV absorbance of quercetin oxide was measured (in the range of 250 nm to 330 nm), and the absorbance of Fe(III) was obtained by plotting the optical density relative to the wavelength.

[0378] Based on the spectrum in the 250nm–330nm region, the area under the curve (AUC) between 285nm and 305nm is calculated using the following formula:

[0379]

[0380] Therefore, using Fe 3+ Each point in the range was plotted relative to each area of ​​the surface from the peak 285nm to 305nm. Trend curves were plotted (Excel), and the correlation coefficient R (the closest value of which is 1) was derived. 2 The experiment was verified (Excel).

[0381] It can be in linear or polynomial form: y = ax 2 +bx+c. Through experiments, this relationship can be expressed as y=-0.0021x 2 +0.4819x+0.4521; R2=0.9966( Figure 2 (4)

[0382] For the mixture according to the invention, in plasma medium or by Fe 2+ The generated Fe 3+ The concentration of the equivalence reflects the oxidation potential of the present invention and is calculated by equation (4). The concentration of Fe was determined under UV light. 3+ Oxidized quercetin is an indirect method for measuring the redox power of mixture 1. Therefore, Fe is mentioned. 3+ The concentration of the "equivalent". Moreover, the redox potential of the solution of the present invention to be tested can be compared with respect to the reference solution according to the present invention; in other words, the redox potency of the mixture according to the present invention can be quantitatively compared.

[0383] 2-Fe 2+ Absorption diagram

[0384] The generated Fe(II) ions were plotted in the same manner as Fe(III) ions. For example, measurements were taken within the above range using a 1.22 μM active material (250 μg / L) and Fe... 2+ The Fe obtained by contacting the mixture of the present invention for 2 minutes 3+ The UV absorbance (230 nm–500 nm) of the oxidized quercetin peak. The same AUC mathematical analysis can be performed on Fe(III) ions as described above.

[0385] 3- The redox potential of the inventive mixtures, for example with 1.22 μM (250 μg / L) of the active substance MoO4 2 - was determined. force.

[0386] According to the steps described in B-, the mixture according to the invention (1.22 μM MoO4 in human plasma) was tested. 2 -) Redox potency: i) assess its aging at room temperature and 45°C, ii) validate its production and comparatively evaluate the formulation, iii) quantify its transdermal pathway, iv) its bioavailability, and v) its biophase.

[0387] According to equation (4), by mixing the mixture of the present invention with transferrin Fe at a final concentration of 1.22 μM... 3+ Free Fe is generated after the complex plasma is exposed for 2 minutes. 3+ The specific surface area (as AUC) of the UV absorption peaks of oxidized quercetin (equations (1), (2), and (3)) between 285 and 305 nm was calculated. On average, the generated Fe... 3+ Equivalent (Fe) 3+ eq The concentration is approximately 30 μg / ml to 50 μg / ml of plasma.

[0388] 4- Estimated curve of effective concentration of the mixture according to the present invention in plasma medium

[0389] Plasma iron is not free. Instead, it exists as ferric iron (Fe3+) in amounts of 1-2 residues per molecule. 3+ It binds to transferrin (or lateral transferrin). Aside from its complexation with hemoglobin, ferrous iron (Fe2+) is not present in its ferrous form. 2+ The circulation was considered to be non-hemolytic in the human plasma used. The quercetin reaction of the mixture of the present invention with plasma copper was considered negligible.

[0390] A series of concentrations of the mixture of the present invention (final MoO4) were incubated in human plasma. 2- The concentrations were 15.2 nM, 7.6 nM, 3.8 nM, and 1.9 nM. The reaction with quercetin was carried out as described in B-1. The specific absorption peak (minus plasma blank + quercetin) was integrated as described in B-1. Figure 3 ).

[0391] Plot a curve based on the polynomial (Excel) and calculate the trend equation (y = 0.0144x). 2 +0.5863x+0.4511, where R 2 =0.9934, (5); Figure 4 ).

[0392] Based on the aforementioned range of the mixture of the present invention, the proportional relationship between the concentration of the present invention added to human plasma and the amount of Fe3+ equivalent detected by the oxidation reaction of quercetin (Equation 4) was determined (Equation 5), namely 28.95 μg Fe 3 + eq / mL human plasma, 12.19μg Fe 3+ eq / mL human plasma, 3.97μg Fe 3+ eq / mL human plasma, 3.12μg Fe 3+ eq / mL human plasma.

[0393] Example 5 - Evaluate the redox efficacy of the present invention on human plasma.

[0394] The mixture according to the invention (Example 1 - "Mixture 1") was added to human plasma and, after a 2-minute contact time, a MoO4 concentration of 0.03038 μM was achieved. 2- Concentration, derived from plasma transferrin-Fe 3+ The complex and Fe produced by the Fenton-Haber-Weiss reaction 3+ To optimize or linearize the oxidation reaction of quercetin (see Example 4).

[0395] After subtracting the experimental blank, the increase in the concentration of the present invention will be due to the shift in the Fenton-Haber-Wes reaction equilibrium. And the in-situ generation of Fe 3+ Decrease in oxidized quercetin. MoO4 2- The final tested concentrations were 0.00 μM, 1.22 μM, 4.05 μM, 12.15 μM, 24.30 μM, and 30.38 μM. Figure 5 ).

[0396] Example 6- Anti-replication efficacy

[0397] A -Model

[0398] Four contact models of the mixture according to Example 1 (“Mixture 1”) were tested. They represent four physiological possibilities that Mixture 1 might encounter in a topical therapeutic application, namely:

[0399] Model 1: Mixture 1 comes into contact with cells infected with HSV-1;

[0400] Model 2: Mixture 1 with Mixture 1 on cells not yet infected with HSV-1;

[0401] Model 3: Mixture 1 comes into contact with uninfected cells and mixes come into contact with free HSV-1 (non-infectious);

[0402] Model 4: Mixture 1 is exposed to pre-infected HSV-1.

[0403] Mixture 1 was tested with two contact times, either 2 minutes or 1.5 minutes, and then mixture 1 was removed.

[0404] Two concentrations of the active substance were tested: 0.81 μM and 2.03 μM, which are 167 μg / L and 417 μg / L, respectively.

[0405] The method for quantifying efficacy is qPCR (quantitative polymerase chain reaction; Mullis K. et al. (1986), “Specific Enzymatic Amplification of DNA In Vitro: The Polymerase Chain Reaction”, Cold Spring Harb. Symp. Quant. Biol., 51(Pt 1), 263-273).

[0406] The anti-replication efficacy (AE) of Mixture 1 against HSV1 was evaluated in qPCR using virus genome-specific primers (Kessler HHe et al. (2000) "Detection of Herpes simplex virus DNA by real-time PCR" J. Clin. Microbiol., 38(7), 2638-2642).

[0407] The cytotoxicity of mixture 1 to the BHK-21 line was assessed in qPCR using a gene-specific primer for the small ribosomal subunit 18S (Texcell-Evry, France).

[0408] Multiplicity of Infection (MOI): 1, or 5.10 5 TCID 50 (50% of the tissue culture infection dose).

[0409] qPCR experiments were performed at t0h (2 hours after exposure to BHK-21 and HSV-1, followed by removal of HSV-1), t2h, t4h, and t8h post-infection.

[0410] The AE / C ratio provides the in vitro potency index (EI) of mixture 1 corresponding to the study model.

[0411] Model 1:

[0412] BHK-21 + HSV-1 (2h; infection) => Wash (PBS) to remove virus => Sampling, t0h => + Mixture 1 (2 min) => Wash (culture medium) to remove Mixture 1 => Incubate cells at 37°C => Sampling at t2h, t4h and t8h post-infection.

[0413] Model 2:

[0414] BHK-21 + Mixture 1 (1.5 min and 2 min) => Wash (PBS) to remove Mixture 1 => Cells + HSV-1 (2 h; infection) => Wash (culture medium) to remove HSV-1 => Sampling, t0 h => Incubate cells at 37 °C => Sampling at t2 h, t4 h and t8 h post-infection.

[0415] Model 3:

[0416] BHK-21+ mixture 1 (1.5 min and 2 min) => Wash (PBS) to remove mixture 1.

[0417] HSV-1+ mixture 1 (1.5 min and 2 min) => Wash (PBS) to remove mixture 1.

[0418] Mix the cells and virus that have been pre-contaminated with mixture 1 (2h; infection) => Wash (culture medium) to remove HSV-1 => Take samples at t0h => Incubate cells at 37°C => Take samples at t2h, t4h and t8h post-infection.

[0419] Model 4:

[0420] HSV-1 + Mixture 1 (2 min) => Wash (PBS) to remove Mixture 1 => + BHK-21 (2 h; infection) => Wash (culture medium) to remove HSV-1 => Sampling, t0 h => Incubate cells at 37 °C => Sampling at t2 h, t4 h and t8 h post-infection.

[0421] B- result

[0422] Table 2 : After 8 hours of contact with mixture 1 (based on μg / L active substance) or acyclovir (ACV), the proliferation of HSV-1 in BHK-21 cells was reduced; in vitro potency index (EI).

[0423]

[0424] * The reduction in HSV replication (RR) induced by the test substance (mixture 1, control acyclovir) is expressed as a percentage and corresponds to the qPCR quantitative ratio between the HSV-1 genome and cytotoxicity assessed by quantifying the host cell subunit 18S: RR = {(100 × ([HSV-1])} 测试 [18S] 测试 )) / ([HSV-1] 对照+ [18S] 对照+ )}100.

[0425] **The in vitro anti-replication potency index (IE) of the test substance was determined by the [HSV-1] / [18S] ratio in specific qPCR of the HSV-infected positive control, i.e., ([HSV-1] / [18S]). 对照+ [18S] 对照+ [HSV-1] of the tested substance (mixture 1, control acyclovir) 测试 [18S] 测试 The ratio is expressed as: IE = ([HSV-1]) 对照+ [18S] 对照+ ) / ([HSV-1] 测试 [18S] 测试 )

[0426] #[ACV]: 1g / L

[0427] The optimal EI was obtained at a contact time of 2 minutes and a concentration of 417 μg / L for mixture 1 (Table 2). It was found that the contact time had a significant effect on the EI (1.5 minutes and 2 minutes; Model 2 and Model 3).

[0428] When the contact time was 2 minutes and the concentration of mixture 1 was 167 μg / L, the EI obtained by model 1 and model 3 was extremely significant.

[0429] Regardless of the model, the EI obtained with acyclovir (“ACV”) was not significant (Table 2).

[0430] surface 3: Quantify i) the proliferation percentage of BHK-21 line (control: uninfected line) and ii) the replication percentage of HSV-1 (control: infected line) by qPCR (18S or HSV gene; 2 minutes after exposure and 8 hours after exposure).

[0431]

[0432] For mixture 1 (167 μg / L~417 μg / L) or ACV (1 mg / L or 10 mg / L) 3Regardless of the control used (lineage + PBS-HSV or lineage + PBS + HSV), the quantification results of 18S were comparable for the same model (Table 3, Test 1 vs. Test 4, Test 2 vs. Test 5, Test 3 vs. Test 6).

[0433] For mixture 1, the antireplication efficacy against models 1, 2, and 3 was confirmed to be significantly dose-dependent (Table 3, Tests 7 and 8). This was not observed for ACV (Table 3, Test 9).

[0434] Based on Model 2 and Model 3 (Table 3, Test 1, Test 3, Test 4 and Test 6), mixture 1 (167 μg / L) and ACV (1 mg / L) (Table 3) were found to have comparable cytotoxicity.

[0435] ACV (1 mg / L or 1 g / L) was found to have no anti-HSV efficacy (Tables 2 and 3, Test 9). The model introduced for studying mixture 1 does not correspond to studies related to ACV.

[0436] For Model 1 (mixture 1 in contact with infected cells):

[0437] The optimal EI for the two concentrations of mixture 1 (2-minute contact, 167 μg / L and 417 μg / L, EI: 37 and 48 respectively) (Table 2).

[0438] Mixture 1 may have a direct effect on intranuclear viral replication and on cellular metabolism (Tables 2 and 3).

[0439] Mixture 1 showed good specificity for infected cells (Table 3, Tests 4 and 5; Model 1 vs. Model 2).

[0440] For Model 2 (mixture 1 in contact with pre-infection cells):

[0441] An interesting EI (Table 2; contact time 2 min and 417 μg / L, EI: 23).

[0442] Mixture 1 works on cells, making them less susceptible to infection and / or acquiring metabolisms incompatible with viral replication (Table 3, Test 7 and Test 8).

[0443] For Model 3 (mixture 1 was individually contacted with uninfected cells and HSV-1 before mixing), ):

[0444] Interesting EIs at the two concentrations of mixture 1 (2-minute contact, 167 μg / L and 417 μg / L, EI: 14 and 35, respectively) (Table 2).

[0445] Depending on the application dose (167 μg / L or 417 μg / L), it exhibits either zero cytotoxicity (Table 3, Test 7 and Test 8, respectively) or limited cytotoxicity (Table 3, Test 4) for very good antiviral efficacy (Table 3, Test 5).

[0446] In this paper, the potency of mixture 1 is the superposition of model 2, which has a significant effect on cells alone, and model 4, which has a limited effect on viruses (Tables 2 and 3).

[0447] For Model 4 (mixture 1 in contact with HSV-1):

[0448] Low EI (exposure time: 2 minutes, 167 μg / L and 417 μg / L, EI: 2 and 3 respectively; Table 2), which was confirmed by the absence of cytotoxicity but significant viral replication (Table 3, Test 8 and Test 9).

[0449] C- in conclusion

[0450] The main effects of mixture 1 on the BHK-21 line are: i) preferential cytotoxicity against infected cells; ii) inhibition of viral replication in infected cells; and iii) restriction of infection in healthy cells.

[0451] The targets can be membranes (e.g., viral receptors) and / or metabolism (e.g., regulation of stress and apoptosis pathways).

[0452] Virus identification and internalization

[0453] According to Model 4, Mixture 1 exhibits low direct anti-replication potency against the virus. Under these experimental conditions, which are close to those for local therapeutic use, this observation is favorable for limiting the alteration of viral receptors to the following cells: gB (HSV-2), gC (HSV-1), gD, and gH / gL (HSV-1 and HSV-2). Mixture 1 alters viral glycoproteins only in a limited manner through its in vitro Fenton-Haber-Wes reaction.

[0454] Similarly, no significant effect was found on mixture 1 on HSV phospholipid membranes that might interfere with membrane fusion and penetration.

[0455] Under these experimental conditions, mixture 1 was not a virus-killing agent.

[0456] Viral replication and cellular metabolism:

[0457] qPCR was performed using cell lysates 8 hours post-infection. The reduced viral replication may be due to poorer recognition and / or internalization and / or replication (alterations in cellular metabolism caused by infection and / or mixture 1).

[0458] If, regardless of the model, mixture 1 alters the viral desalination pathway without altering other pathways, the resulting EI will be smaller due to the accumulation of viral cells in the cytoplasm.

[0459] According to Model 2 (EI: 23, 2 minutes of contact time and 417 μg / L), low viral replication was observed after cells were pre-contaminated with mixture 1 (Tables 2 and 3, Test 8).

[0460] According to this model, BHK-21 cells were exposed to Mixture 1 for 2 minutes and then exposed to HSV in a complete nutrient medium for 2 hours. Mixture 1 induced cellular changes for more than two hours, which further demonstrates the preventive efficacy of Mixture 1.

[0461] In addition to recognizing HSV-1 using cells partially affected by mixture 1 (which has a possible and limited effect on viral glycoprotein gD (38% replication; Table 2)), this recognition may also be influenced by the effects of cell receptors on gD (HVEM and integrin) and by the effects of two other surface cell receptors on gB and gH / gL (heparin sulfate and integrin, respectively).

[0462] Free radical permeation (altering intracellular or transmembrane redox potential, regulating oxidative stress pathways) can alter cellular calcium influx. This cellular calcium influx is also induced by HSV infection. However, the calcium flow induced by HSV infection can be immediately stopped by the introduction of free and non-permeable chelating agents into mixture 1 (limiting cellular cytotoxicity). On the other hand, other major cellular alterations should not be ruled out, and may correspond to the oxidation or reduction of proteins (e.g., cysteine ​​bridges), particularly the oxidation or reduction of structural proteins that may occur during viral endocytosis.

[0463] According to Model 3 (Tables 2 and 3, Test 7 and Test 8), a significant reduction in viral replication was observed (exposure time 2 minutes, 167 μg / L and 417 μg / L, EI: 14 and 35, respectively).

[0464] Both phenomena are cumulative. The first is a limited effect on viruses (see Model 4), while the second, more significant effect is on cells (see Model 2).

[0465] The sum of the EIs (i.e. 23 and 3 respectively) of Model 2 and Model 4 (2 minutes and 417 μg / L) is (26) less than but close to the experimental value of Model 3, i.e. 35.

[0466] Imagine potential accumulation of: i) poor recognition of HSV couples and cells and ii) internalization and / or replication, particularly due to alterations in cellular metabolism.

[0467] Model 1 (EI: 48, contact time of 2 minutes, 417 μg / L) was the most effective.

[0468] The anti-infective effect of mixture 1 is preferably exerted on cells rather than on viruses. This effect is more effective because the cells are pre-infected.

[0469] In this model corresponding to the optimal treatment problem, mixture 1 clearly exhibits specific intracellular pharmacological potency. Table 3 shows that for 417 μg / L of active substance, the amount of ribosomal subunit 18S in this model (27% and 30% according to the controls used, Test 2 and Test 5) is lower than that in model 2 (41% and 44% according to the controls used, Test 2 and Test 5) and model 3 (70% and 58% according to the controls used, Test 2 and Test 5), providing evidence of specificity relative to healthy cells for infected cells.

[0470] In this model, the target of mixture 1 is neither identifiable nor internalized, but rather a metabolic alteration. The latter is in a new equilibrium state, particularly due to changes in free radical transmembrane channels, alterations in cellular redox potential and the presence of external chelators, oxidative stress pathways (already induced by infection), calcium flux (already induced by infection), inhibition of calcium-dependent release from the virion, and oxidative modifications of proteins directly or indirectly involved in viral replication.

[0471] Example 7 - In vitro assessment of interleukin production

[0472] Non-pathological biopsy tissues (8 biopsy tissues and 4 donors) obtained from weight-loss surgery were ex vivo processed with mixture 1 (5 mg / L active substance; 2 min). Biopsy tissues were collected and sampled from the supernatant at 6 hours and 24 hours after exposure to quantify interferon-α (IFN-α; inhibits HSV infection; Mikloska Z. et al (2001) "Alpha and Gamma Interferons Inhibit Herpes Simplex Virus Type 1 Infection and Spreadin Epidermal Cells after Axonal Transmission", J.Virol., 75(23), 11821-11826), interferon-β (IFN-β; inhibits HSV replication; Sainz Jr. B. et al (2002) "Alpha / Beta Interferon and Gamma Interferon Synergize To Inhibit the Replication of Herpes Simplex Virus Type 1", J.Virol., 76(22), 11541-11550) and interleukin-6 (IL-6; inflammatory marker).

[0473] The positive control was physiological saline (100mM NaCl).

[0474] No changes were observed in the basal cellular levels of IFN-α and IFN-β.

[0475] Based on the biopsy tissue and donor, and based on the trauma from surgical and experimental procedures:

[0476] 6 hours after exposure ( Figure 6 and Figure 7 ):

[0477] NaCl was used as a control, producing baseline IL-6 levels ranging from 277 pg / mL ± 11 pg / mL to 2,866 pg / mL ± 206 pg / mL.

[0478] Using mixture 1, relative to the NaCl control, the production of IL-6 was inhibited from 0 pg / mL to 1,015 pg / mL ± 17.

[0479] 24 hours after contact ( Figure 6 and Figure 7 ):

[0480] NaCl was used as a control, and the baseline IL-6 levels ranged from 1485 pg / mL ± 37 pg / mL to 7454 pg / mL ± 199 pg / mL.

[0481] Using mixture 1, relative to the NaCl control, the production of IL-6 was inhibited from 84 pg / mL to 5,910 pg / mL ± 29 pg / mL.

[0482] in conclusion:

[0483] Following contact with NaCl and mixture 1 (6 hours and 24 hours, respectively), IL-6 was produced unevenly and excessively. Figure 6 ).

[0484] Compared to the NaCl control, after contact with mixture 1 (5 mg / L active substance), the inhibition of IL-6 production was uneven, averaging 28% at 6 hours and 19% at 24 hours. Figure 6 ).

[0485] Through this inhibition of production, mixture 1 possesses anti-inflammatory properties. Figure 7 ).

[0486] Example 8 - Plasma Degradation and Half-Life

[0487] For the active material MoO4 in mixture 1 2- Fenton-Haber-Western type reactions, free radicals (mainly HO2) · One of the donors for this tracer (which has a lifespan of several seconds and a significant cell penetration index) is H2O2. The degradation of this tracer was tracked to assess the consumption rate of mixture 1 or its inactivation.

[0488] technology:

[0489] Mixture 1 (final concentration 68.9 μM or 14.2 μg / L) was incubated in fresh human plasma. The kinetics of H2O2 disappearance were monitored by peroxidase reaction on aliquots sampled at continuous time intervals.

[0490] result:

[0491] After a 2-minute contact time, at room temperature, the average (n=10) percentage degradation of the tracer of mixture 1 in human plasma was 85.3% ± 9.4%, of which it was 74% ± 15.2% in the first minute.

[0492] Within the first minute, for a solution of a mixture 1 containing an initial concentration of 68.9 μM of active material and an initial concentration of 1 mM of H2O2, the "degradation time 50%" or DT 50It takes 0.812 minutes.

[0493] The degradation rate K is within the first minute of incubation at room temperature. cH2O2 ≈ 740 μmol of mixture 1, or K in an initial 1 mM H2O2 / min / L plasma cMoO42- ≈(Initial 68.9 μM active material MoO4) 2- 1. A mixture of 51.0 μmol / L plasma at a concentration of 1 / min / L.

[0494] For a 200 μL sample of Mixture 1, 24.3 μM (5.0 mg / L) of the active substance or 353 mM (12 g / L) of the tracer H2O2, when applied topically for therapeutic purposes, has approximately 5 nmol of active substance MoO4. 2- Alternatively, 70 μmol of tracer H₂O₂ can be used. The sample degrades in 1 L of plasma within approximately 0.1 minutes.

[0495] Example 9 - Limited hematologic toxicity to peripheral blood.

[0496] The aim of this study was to evaluate the hemotoxicity of mixture 1 after incubation in human peripheral blood for 2, 3, and 5 minutes (final concentration 24.3 μM or 5.0 mg / L). Hemolysis was assessed based on key hematologic parameters: red blood cell count, white blood cell count, hematocrit, platelet count, mean globulin volume, and hemoglobin level.

[0497] in conclusion:

[0498] Hematological parameters related to red blood cell count, white blood cell count, hematocrit, mean globulus volume, and hemoglobin level will not be altered by incubating the mixture of the present invention for up to 5 minutes.

[0499] Upon contact with Mixture 1, the platelet count immediately changed from -35% to -69% within 5 minutes after the addition of Mixture 1.

[0500] Example 10 - Limited transdermal pathways, absorption, biophase and bioavailability of the mixture according to the invention (OECD 428; EMEA, Human Guideline, 2001).

[0501] Mixture 1 was concentrated 5-fold (mixture 1 × 5) to reveal the minimum channels of the substance. The aim of this study was to evaluate (referencing t0, t2 min… to t60 min) the channels of mixture 1 × 5 and the channels of tracer H2O2 through non-pathological human skin biopsy tissue.

[0502] This method is applicable to three types of substrates: all biopsy tissues, biopsy tissues cut in the epidermis, and biopsy tissues cut in the dermis.

[0503] technology:

[0504] Twelve biopsy tissues from two donors were tested according to two methods (Table 4) (measured in a droplet on a skin flap [30 μL] and in a culture medium [800 μL] below it): i) assessing the redox potency of quercetin 1 × 5 and ii) measuring the concentration of peroxidase in tracer H2O2.

[0505] In droplets (30 μL) placed above biopsy tissue and epidermal and dermal flaps, the concentrations of the active substance (121.5 μM or 25.02 mg / L) and tracer (1.77 M or 60 g / L) were 5-fold concentrated relative to the concentrations of the therapeutic agents (24.3 μM or 5.0 mg / L and 352.8 mM 12 g / L, respectively). In a 1 × 5 deposit of the 30 μL mixture, there were 3.65 nmol or 750 ng of active substance and 53 μmol or 1.8 mg of tracer.

[0506] result:

[0507] Table 4

[0508]

[0509] * Relative to t0, expressed as % (-: decrease, +: increase)

[0510] **Detection limit.

[0511] A decrease in the redox potency of mixture 1 was observed in the droplet above the skin flap, ranging from low to moderate (Test 1) (from -1.4% to -23.9%).

[0512] A “moderate to significant” decrease in the amount of tracer H2O2 in mixture 1 was observed in the droplet above the skin flap (test 2) (from -16.5% to -99.9%).

[0513] An increase in the redox potency of the subcutaneous medium was observed from low to high (Test 3). This depends on the flap and its type. When the tissue is intact biopsy tissue, this increase due to the percutaneous access is low. It averages 6.35%, equivalent to passing through a mixture of 232 pmol (47.6 ng) of active material¹. When the tissue is epidermis or dermis, this increase varies considerably (from 0.8% to 100%). This is likely due to differences in histological structures such as pores, skin blood perfusion, and epidermal nerves.

[0514] Regardless of the type, an increase in the amount of tracer H2O2 in the subcutaneous medium was observed from undetectable to slight (Test 4) (from <+0.001% to 1.7%). This increase was particularly low in intact biopsy tissue due to the percutaneous access (<0.53 nmol to 47.7 nmol, i.e., <0.018 μg to 1.62 μg). In the epidermis or dermis, this increase was on average small (1.04% and 0.31%, i.e., 0.55 μmol and 0.16 μmol, or 18.7 μg and 5.6 μg, respectively).

[0515] in conclusion:

[0516] Two parameters representing tissue independence (absorption, inactivation, permeation) that are considered typical characteristics of mixture 1 exist: the redox potency of the active substance and the dosage of the peroxide tracer (free radical donor).

[0517] Considering the entire biopsy tissue, the penetration index of mixture 1 corresponds to the thickness of the flap, i.e., 1 mm (average thickness of the epidermis) to 2 mm.

[0518] The epidermis contains numerous nerve endings, which may be central to axons, allowing the patient's herpesvirus bodies to desalinate. These are the same targets of Mixture 1 as the infected epidermal cells or those prior to potential infection. Considering the continuity of the two tissues, the biological phase of Mixture 1 is almost strictly one epidermal thickness (1 mm) and generally achieves maximum absorption of the target tissue.

[0519] Given that the dermis alone is vascularized, it can be re-examined that the redox potency of Mixture 1 upon contact is at most 80.6% and that of the tracer at most 1.7%. Under these experimental conditions, the bioavailability of Mixture 1 × 5 is 2.94 nmol or 605 ng of the active substance and 0.9 μmol or 30.6 μg of the peroxide tracer.

[0520] In in vitro genotoxicity studies, the mixture and its metabolites according to the invention were not mutagenic in the presence of activator S9 (5 / 5 Salmonella strains) by the Ames test, but were mutagenic to 1 / 5 strains in the presence of activator S9.

[0521] When applied topically to the skin (irritating or non-irritating), the mixtures according to the invention (24.3 μM or 5.0 mg / L active substance) do not exhibit any associated irritant toxicity.

[0522] In oral and vaginal irritation studies, the mixtures according to the invention (24.3 μM or 5.0 mg / L) were irritating (score 0 / 16) and very irritating (score 1 / 16), respectively.

[0523] In studies of highly sensitive chorioallantoic membrane stimulation in chicken embryos, mixture 1 (18.23 μM and 24.3 μM or 3.75 mg / L and 5.0 mg / L) was moderately irritating (irritation index or IS 6.8 ± 0.4 and 6.3 ± 0 / 21, respectively).

[0524] In acute toxicity studies conducted via dermal application, the mixture of the present invention was well tolerated without any symptoms at doses of 25.0 μg / kg and 31.3 μg / kg of active substance in 60 mg / kg and 75 mg / kg of H2O2 tracer, respectively, representing the "Maximum Tolerable Dose" (MTD) and the "Maximum Dose with No Observed Adverse Effects" (MDWNODE); or at 31.3 μg / kg of active substance and 75 mg / kg of tracer, representing the "No Observed Adverse Effect Level" (NOAEL).

[0525] In acute toxicity studies conducted via intravenous injection, the mixture of the present invention was tolerated without any significant symptoms at doses of 10.4 μg / kg and 12.5 μg / kg of active substance in 25 mg / kg and 30 mg / kg of H2O2 tracer, respectively. The "maximum tolerated dose" (MTD) was 12.5 μg / kg of active substance.

[0526] Example 11 - Acceptable toxicity of the mixture according to the invention

[0527] I- In vitro mutagenicity (Ames test - OECD 471)

[0528] The aim of this study was to evaluate the mutagenic activity of the active mixture (mixture 1) according to the invention and its metabolites (produced from the S9 portion of mouse liver) against Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535.

[0529] in conclusion:

[0530] In the absence of any S9 activation system, the active mixtures (7.7 μM or 2.43 μM, i.e. 1.58 mg / L or 0.50 mg / L) and their metabolites according to the invention have no mutagenic effects on the TA97a, TA98, TA100 and TA1535 lines.

[0531] In the presence of the S9 activation system, the active mixture (mixture 1×5: 121.5 μM or 25.02 mg / L) and its metabolites according to the present invention have no mutagenic effect on the TA97a, TA98, TA100, TA102 and TA1535 lines.

[0532] In the absence of the S9 activation system, the active mixture according to the invention is not mutagenic to strain TA102 at 0.50 mg / L.

[0533] In this in vitro test, the mutagenicity of the active mixture and its metabolites according to the invention was limited for their use (strain TA102).

[0534] II- Induced skin sensitization (female hamsters; OECD 406, ISO 10993-10:2013 and ICH memorandum) SCCP2005)

[0535] Based on the schemes of Magnusson and Kligman (Magnusson B. et al. (1969) "The identification of contact allergens by animal assay. The guinea pig maximization test", J. Invest. Dermatol., 52(3), 268-276), the sensitization potential of female hamster skin was studied using three schemes.

[0536] Option 1: Determine the tolerance limit dose

[0537] A mixture containing 1 mL of H2O2 tracer active material at a concentration of 1.25 mg / L to 20.00 mg / L, in which 3 g / L to 48 g / L, is applied to the flank of an animal and left for 24 h, after which the animal is observed.

[0538] Mixture 1, containing 10 mg / L of active substance in 24 g / L tracer, exhibited good tolerability and resulted in moderate irritation.

[0539] The concentration of 10 mg / L of active substance contained in 12 g / L tracer in mixture 1 was well tolerated and considered to be the maximum non-irritating dose.

[0540] Option 2: Determine the tolerance limit dose after subcutaneous injection induction.

[0541] A mixture containing 5 mg / L to 10 mg / L of active substance in an H2O2 tracer at a concentration of 12 g / L to 24 g / L was injected subcutaneously (100 μL). At day 8 (first treatment, challenge), a 1 mL patch was applied topically for the first time. At day 27 (second treatment), a 1 mL patch was applied topically for the second time. Observations were conducted on day 29.

[0542] The concentration of 5 mg / L active substance in Mixture 1, containing 12 g / L tracer, was well tolerated and did not produce any irritation.

[0543] Option 3: Research by The irritation caused by mixture 1 containing 5 mg / L of active substance in 12 g / L H2O2 tracer and its topical application.

[0544] 100 μl of 5.0 mg / L Mixture 1 was injected subcutaneously. On day 4, the injection site was stimulated with a 10% sodium dodecyl sulfate (SDS) solution, followed by a topical application of Mixture 1 (1 mL) in patch form. A second topical application was completed on day 6. On day 21 (treatment), a topical application of Mixture 1 (1 mL) in patch form was completed, wherein the concentration of Mixture 1 was 6.26 mg / L to 10 mg / L of active substance contained in a tracer at a concentration of 15 g / L to 24 g / L.

[0545] The mixture 1 containing 5.0 mg / L of active substance in 12 g / L tracer was well tolerated, and the skin sensitization score of the preparation was 0 / 3.

[0546] in conclusion: The mixture 1, containing 5.0 mg / L of the active substance, was non-irritating. No macroscopic changes were observed in the animal skin. Therefore, no anatomical or pathological studies were necessary.

[0547] III- Oral and vaginal stimulation (female rabbits; OECD(99)20, -21, -23, -24, -(95)115, -(02)9 and ISO- 10993-10:2013)

[0548] The aim of this study was to evaluate the vaginal and oral tolerability of mixture 1 containing 5.0 g / L of active substance in 12 g / L tracer when exposed for 5 days for human therapeutic use.

[0549] From day 0 to day 6, administer 1 mL of the solution to the animals every 24 hours via oral and vaginal routes. On day 7, observe the animals and measure their weight.

[0550] in conclusion Mixture 1, with an active substance concentration of 5.0 mg / L, exhibited good tolerability. The oral and vaginal irritation indices were 0 / 16 and 1 / 16, respectively. Therefore, no anatomical pathological studies were necessary.

[0551] IV- Eye irritation test (ICCVAM App B)

[0552] The ocular irritation test used was the alternative test HET-CAM (Chicken Embryo Test-Villiloallantoic Membrane), published in the official journal on December 26, 1996. It was recommended by ICCVAM.

[0553] Although this particularly sensitive method has not been officially verified by the EU, it is acceptable in identifying even slightly irritating substances, including those that irritate the eyes and are assigned the label R41 (2002).

[0554] In summary, the method involves applying a test substance (a mixture containing 3.75 mg / L and 5.00 mg / L of active substance in 9 g / L and 12 g / L tracers, respectively) to the chorioallantoic membrane of a fertilized egg. After contact, the following three criteria were observed and scored at 0.5 min, 2 min, and 5 min: hemorrhage, clotting, and congestion.

[0555] Results and conclusions:

[0556] A mixture of active substances at 3.75 mg / L or 5.00 mg / L can be classified as moderately irritating (irritation index or IS equal to 6.8±0.4 and 6.3±0 / 21, respectively), meaning that it induces mild cytolysis (severity score = 1 / 3) immediately after introduction or within 30 seconds of exposure, without bleeding or clotting.

[0557] Considering the Q value (the ratio of the exponents of the positive substance reference NaOH 0.1M [IS = 15 ± 3] and 1% SDS [10 ± 2] to mixture 1), for mixture 1 with active substances of 3.75 mg / L and 5.00 mg / L, the Q values ​​relative to NaOH are 0.51 to 0.54 and relative to SDS are 1.10 to 1.18, respectively.

[0558] Therefore, for this extremely sensitive test, which is more significant than the vaginal stimulation test that has been performed efficiently in female rabbits, mixture 1 is considered to be a mild (3.75 mg / L) to moderate (5.00 mg / L) irritant.

[0559] V- Acute toxicity was studied through skin administration and intravenous injection—maximum tolerated dose (MTD) and no observable adverse events. Observed toxicity dose (NOAEL) (rat; OECD 474, ICH M3 (R2) and S6 (R1))

[0560] The aim of this study was to determine the maximum tolerated dose (MTD) of mixture 1 by transdermal administration (1 ml) or intratailed injection (5 ml / kg). Clinical observations were recorded until day 14.

[0561] Conclusions of the skin application regimen:

[0562] Mixture 1, containing 50 μg / kg of the active substance (1 ml to 10 mg / L), was well tolerated. Mild erythema and small crusts were observed in the treated area, which disappeared within 2 days. No irritation was observed.

[0563] For mixture 1, in which the active substance was administered at a concentration of 37.5 μg / kg (1 ml to 7.5 mg / L), the same proportion of milder symptoms was observed.

[0564] The mixture 1, containing doses of 25.0 μg / kg and 31.3 μg / kg of active substance (1 mL, 5.0 mg / L and 6.26 mg / L), was well tolerated. No skin, metabolic or physiological symptoms were observed on day 14.

[0565] In dermal administration to rats, the MTD of mixture 1 was 31.3 μg / kg of active substance (1 mL, 6.26 mg / L).

[0566] In dermal administration to rats, the NOAEL (no observed toxicity) of mixture 1 was 31.3 μg / kg of active substance (1 mL, 6.26 mg / L).

[0567] Conclusion of the intra-tail injection protocol:

[0568] The challenge with intravenous administration of mixture 1 lies not in the inherent toxicity of the invention, but in its decomposition upon contact with blood, particularly the generation of molecular oxygen that can lead to embolism. The ethically limited dose (3 rats) that has been shown to cause reversible respiratory arrest is 14.6 μg / kg of the active substance (5 ml / kg of mixture 1, 2.92 mg / L). No signs of toxicity were recorded up to day 14.

[0569] Following intravenous injection of 12.5 μg / kg of the active substance (5 ml / kg of Mixture 1, 2.5 mg / L), Mixture 1 was well tolerated. Physiological distress (dyspnea and dyspnea) was observed in the animals at the time of injection. No signs of toxicity were recorded until day 14.

[0570] Following intravenous injection of 10.4 μg / kg of the active substance (5 ml / kg of a mixture with a concentration of 2.1 mg / L 1), a similarly proportionate, less pronounced symptom response was observed, with no clinical signs or changes in body weight. No signs of toxicity were recorded until day 14. On day 14, a post-mortem examination of the organs revealed no new problems.

[0571] In intra-tail injection, the MTD of mixture 1 is 12.5 μg / kg of active substance (5 ml / kg of mixture 1 with a concentration of 2.5 mg / L).

[0572] VI- In vivo genotoxicity (male and female) was investigated by examining the generated micronuclei after intravenous injection of mixture 1. Rats; OECD 474 and ICH S2(R1))

[0573] Female rats (n=5) and male rats (n=5) were injected with HPLC water carrier (intravenous route), cyclophosphamide positive controls at 5 mg / kg and 10 mg / kg (intravenous route), ethyl methanesulfonate at 100 mg / kg and 150 mg / kg (intraperitoneal route), and a mixture of active substances at 1.46 μg / kg, 4.17 μg / kg and 12.5 μg / kg (intravenous route) (two treatments, 22-26 hours apart; 5 ml / kg).

[0574] Morbidity, mortality, animal weight, and clinical parameters (temperature; skin; hair; eyes; mucous membranes; secretions and excretions; respiratory and neurological function; gait and posture) were recorded. Blood samples were collected from animals 36 to 48 hours after the second treatment and analyzed by flow cytometry (toxicity comparison: reduction in the proportion of immature erythrocytes (CD-71 positive)). The proportion of micronucleated immature erythrocytes (reticulocytes) was assessed based on 4,000 observations per blood sample. 40,000 observations were represented for each rat population (n = 10) for each test substance (Table 5).

[0575] Table 5:

[0576]

[0577] * Animals that died during the first treatment due to embolism caused by the release of molecular oxygen when mixture 1 came into contact with blood.

[0578] **This study shows a statistically significant difference from the control group.

[0579] # Active substance MoO4 2-

[0580] in conclusion: Compared with the control groups (positive and negative), mixture 1 did not produce any genotoxicity in rats (n=10, including 5 male rats and 5 female rats) after intravenous injection inducing micronucleated reticulocytes, ranging from 1.46 μg / kg, 4.17 μg / kg and 12.5 μg / kg of active substance.

[0581] Example 12 - Comparison with other compositions

[0582] This embodiment specifically illustrates the effect of the composition, particularly its redox potential, on the following:

[0583] The role of 1-calcium in the Fenton / Haber-Weiss "like" catalysis of the test formulation.

[0584] 2- Differences exist between the three formulations of the 5.0 mg / L active substance.

[0585] S1: Mixture 1;

[0586] S2: The composition according to US 6660289;

[0587] S3: The composition according to WO / 2010 / 004161.

[0588] Option 1 (Tables 6 and 7):

[0589] Two parameters were measured: i) pH, ii) redox potential.

[0590] Catalyst CaCl2 (1M, in H2O): Added to a final concentration of 24mM

[0591] Kinetics: Initial (T0), 1 minute, 2 minutes, and 3 minutes after adding CaCl2 with stirring (T1, T2, T3).

[0592] Results and Discussion:

[0593] Table 6

[0594]

[0595]

[0596] The pH of the skin is 4.93 + / - 0.45 to 5.12 + / - 0.56. The pH of the vagina is usually around 4.50, and can reach 6.00 during menopause.

[0597] Solution S1 was buffered to approximately the pH of human skin and mucous membranes. This pH remained relatively constant after contact for 3 minutes following the addition of a final concentration of 24 mM CaCl2. A modest increase in E° was observed, which may be partly attributed to the addition of chlorine to the CaCl2.

[0598] Acidic pH enhances iron-mediated cytotoxic lipid peroxidation.

[0599] Solution S2 was unbuffered. Its pH is not very compatible with skin or mucous membrane contact (including the potential disruption of symbiotic flora in therapeutic use). After the addition of the final 24 mM CaCl2, the pH remained relatively constant until 3 minutes of contact. A slightly less mild increase in E° compared to S1 was observed, which may be partly attributed to the addition of chlorine in the CaCl2.

[0600] Solution S3 was unbuffered. Its pH is not well-suited for contact with skin or mucous membranes (including the potential to disrupt the symbiotic flora in therapeutic applications). After the addition of CaCl2 to a final concentration of 24 mM, the pH remained relatively constant until 3 minutes of contact. A significant increase in E° (+50 mV) was observed, which may be partly attributable to the addition of chlorine in the CaCl2, but the difference in potential increase relative to S1 and S2 is likely not due to this single factor.

[0601] For in situ Fe ions, at acidic pH (<2.33), the redox potential is approximately 400 mV and it is in the Fe(III) form, which is the most cytotoxic form. At pH approximately 4.5, the redox potential is approximately 300 mV, and it is in the (II) form of hemoglobin Fe and the (III) form of transferrin-complexed plasma Fe.

[0602] At the pH of solutions S2 and S3, the iron oxide in the form of Fe(OH)3 is highly unfavorable. Therefore, the Fenton-Haber-Weiss reaction is not very efficient.

[0603] Fe(OH)3 / Fe 2+ The potential value for (Fenton type) is: E'° = 1.19 - 0.18 × pH.

[0604] Table 7

[0605] Active substance (mg / L) pH: <![CDATA[E° Fe(OH)3 / Fe2+ (mV) ＊ ]]> <![CDATA[E° 溶液 (mV)]]> S1 5.0 4.70 344 354 S2 5.0 2.80 686 428 S3 5.0 3.00 650 418

[0606] *For in situ cellular level of iron

[0607] According to this study, Fe(OH)3 / Fe 2+ This is detrimental. Even with improvements to the redox couple (418–428 mV), it remains too significant for cell contact.

[0608] Animal cells have a negative transmembrane potential (≈-70mV). The higher the redox potential of the solution, the greater the change in transmembrane potential and the greater the induction of cellular oxidative stress (apoptosis, gene induction of the eNOS pathway, etc.), while also inducing calcium influx. This may make the significant cellular stress already induced by solutions 2 and 3 potentially become a reality.

[0609] Therefore, the pH and redox potential of formulation S2, and especially the redox potential of formulation S3, are most likely to be cytotoxic. Neither solution S2 nor solution S3 is suitable for therapeutic use.

[0610] Option 2 (Table 8):

[0611] The cellular and plasma concentrations of calcium are approximately 2 mM to 3 mM. This regimen is based on the therapeutic use of the formulation.

[0612] One parameter to be measured: residual amount of peroxide (H2O2, peracetic acid; Quantofix test range between 0 and 25 mg / L).

[0613] Catalyst CaCl2 (1M, in H2O): added at final concentrations of 1.25mM, 2.5mM, 10mM, 25mM and 50mM.

[0614] Final volume: 1 ml = 0.950 μL of test solution + 0.05 μL of CaCl2 diluent. Dilution factor of the test solution: 1.05. The final concentration of the test solution is 11.4 g / L (initial concentration 12 g / L) of tracer H2O2, containing 4.76 mg / L (initial concentration 5.00 mg / L) of active substance.

[0615] Kinetics: Add CaCl2 with stirring for 5 minutes.

[0616] result:

[0617] Table 8

[0618] test Test solution <![CDATA[Final [CaCl2] (mM)]]> <![CDATA[[Peroxide] 终 (mg / L)]]> 1 S1 50 ＞＞25 2 S1 25 10-25 3 S1 10 10 4 S1 2.5 5-10 5 S1 1.25 H25 6 S2 2.5 ＞25 7 S3 2.5 ＞25 8 S3 1.25 H25

[0619] When exposed to 2.5 mM (physiological concentration) CaCl2 for 5 minutes, the inactivation of formulation S1 corresponds to a 1500-fold decrease in peroxides (Test 4), i.e., the tracer decreased from 12 g / L to 8 mg / L and the active substance decreased from 5.00 mg / L to 3.33 μg / L.

[0620] Comparative conclusions regarding formulations S2 and S3

[0621] Regardless of the metal used, solution S2 is incompatible for therapeutic use due to the following:

[0622] For pH values ​​that maintain potential and are not buffered;

[0623] Redox potential of cytotoxicity;

[0624] The relative independence of calcium catalysis;

[0625] Under physiological conditions, it exhibits cytotoxicity due to H2O2 and peracetic acid (except for Ag). + The presence of ions (e.g., those with irreversible cytotoxicity);

[0626] Under the experimental conditions used, there was no Fenton-Haber-Weiss-like catalytic reaction, and therefore no reactive free radicals were generated for therapeutic purposes.

[0627] Regardless of the metal used and the chelating pair described in patent application WO 2010 / 004161, solution S3 is incompatible for therapeutic use due to the following:

[0628] pH incompatible with BAPTA;

[0629] Redox potential with cytotoxicity;

[0630] The relative independence of calcium catalysis;

[0631] Under physiological conditions, it exhibits cytotoxicity due to H2O2 and peracetic acid;

[0632] Under the experimental conditions used, there was no Fenton-Haber-Weiss-like catalytic reaction, and therefore no reactive free radicals were generated for therapeutic purposes.

[0633] Conversely, the reaction chemistry model of the formulation S1 according to the invention is applicable because it is destabilized by calcium.

[0634] For formulation S1, select the following to make the formulation compatible with therapeutic use:

[0635] pH;

[0636] Buffer pair (adding a single component of the pair: acetic acid, because it will slightly produce superacetic acid in situ);

[0637] Transition metals and their valences that are compatible or incompatible with Fenton-Haber-Weiss type reactions;

[0638] Chelating agent pair (for Kd in "cascade") Ca2+ Kd Mo and Kd Fe2+ / Fe3+ (They are compatible with each other);

[0639] Non-cellular permeable chelating agents;

[0640] At physiological doses and pH (the importance of selecting chelating agents), the complex of solution S1 is activated in situ by calcium or other existing metal ions, which in particular limits lipid oxidation (small amount of Fe(III)) and the transient generation of permeable active free radicals without accumulation at redox potential.

[0641] Solution S1 degrades very strongly at a physiological calcium concentration of at least 2.5 mM. Decreases (1.25 mM) or increases (from 10 mM to 50 mM) in non-physiological calcium concentrations strongly alter peroxide degradation via Fenton-Haber-Wesley reactions due to equilibrium shifts (law of mass action). The chemical complexes corresponding to solution S1 are perfectly adapted to specific reactions under cellular and plasma calcium stability conditions.

[0642] Example 13 - Example of an active mixture of the present invention containing a combination of Mo and La.

[0643] This embodiment of the composition according to the invention (“Mixture 2”) comprises a molybdenum salt and a lanthanum salt. The composition was prepared according to Table 9, where each component is expressed as an initial concentration:

[0644] Table 9

[0645] <![CDATA[ substance ]]> <![CDATA[ The mixture of the present invention ]]> Sodium molybdate 20.7μM Lanthanum nitrate 11.5μM BAPTA 42μM EGTA 1051.6μM <![CDATA[H2O2]]> 353mM <![CDATA[CH3COOH / CH3COONa]]> 70mM pH (adjusted by NaOH) 4.4～5.0 <![CDATA[E° 氧化还原 ]]> 300mV~420mV

[0646] The composition is referred to as “initial” because it corresponds to the concentration of the added reagent, without taking into account the catalytic process that occurs.

[0647] All components used in the synthesis and the final product were verified by IR-FT spectroscopy.

[0648] Table 10

[0649] <![CDATA[ product ]]> <![CDATA[ purity ]]> <![CDATA[ Molecular formula ]]> <![CDATA[ N℃AS ]]> <![CDATA[ Sodium molybdate dihydrate ]]> <![CDATA[ 98％～103％ ]]> <![CDATA[ Na2MoO4·2H2O ]]> <![CDATA[ 10102-40-6 ]]> <![CDATA[ Lanthanum nitrate hexahydrate ]]> <![CDATA[ ≥99％ ]]> <![CDATA[ La(NO3)3·6H2O ]]> <![CDATA[ 10277-43-7 ]]> <![CDATA[ BAPTA: (1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) ]]> <![CDATA[ ≥98％ ]]> <![CDATA[ C 22 H 24 N2O 10 ]]> <![CDATA[ 85233-19-8 ]]> <![CDATA[ EGTA: (ethylene glycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid) ]]> <![CDATA[ ≥99％ ]]> <![CDATA[ C 14 H 24 N2O 10 ]]> <![CDATA[ 67-42-5 ]]> <![CDATA[ hydrogen peroxide ]]> <![CDATA[ 29％～31％ ]]> <![CDATA[ H2O2 ]]> <![CDATA[ 722-84-1 ]]> <![CDATA[ glacial acetic acid ＊ ]]> <![CDATA[ 99.8％～100.5％ ]]> <![CDATA[ CH3COOH ]]> <![CDATA[ 64-19-7 ]]> <![CDATA[ Sodium acetate trihydrate ]]> <![CDATA[ 99％～101％ ]]> <![CDATA[ C2H3NaO2.3H2O ]]> <![CDATA[ 5010524 ]]> <![CDATA[ Sodium hydroxide ]]> <![CDATA[ 10N ]]> <![CDATA[ NaOH ]]> <![CDATA[ 1310-73-2 <!-- 34 -->]]> <![CDATA[ softened water ]]> <![CDATA[ 1μS ]]> <![CDATA[ H2O ]]> <![CDATA[ 7732-18-5 ]]>

[0650] The active substance is: La(MoO4) 1- Na + (Lanthium peroxide hydrogenated molybdate).

[0651] Molecular weight: 481.81 g / mol.

[0652] Preparation process:

[0653] Except for the modifications described below, the preparation process is the same as that for mixture 1 in Example 1:

[0654] Two SC solutions were prepared:

[0655] Solution A—Na₂MoO₄·2H₂O (Mo(VI) source; 100%) 200 mg (final concentration 2.4 mM)

[0656] Water qsp 340ml

[0657] Stir gently at room temperature

[0658] Solution B—La(NO3)3·6H2O (La(III) source; 100%) 200 mg (final concentration 1.4 mM)

[0659] Water qsp 340ml

[0660] Stir gently at room temperature

[0661] Then, solution A (10 mL) and solution B (10 mL) were gradually introduced into the Si2 solution to prepare the S1 solution of hydrogenated lanthanum peroxide.

[0662] Step (vii): Add chelating agent - prepare S4 solution

[0663] Introduced BAPTA (98.8%) at 20 mg / L;

[0664] Introducing EGTA (99.1%) at 400 mg / L;

[0665] The pH of the final solution was adjusted to 4.67 using NaOH.

[0666] The final E 氧化还原 380mV

[0667] In mixture 2, La(MoO4) 1- Na + The final concentration was 20.8 μM.

[0668] Example 14 - Evaluation of the redox efficacy of the molybdate and lanthanate dual complexes of the present invention on human plasma.

[0669] To compare (MoO4) 2- (Mixture 1) and La(MoO4) 1- The oxidizing power of (mixture 2) formulation makes the concentration similar to... Figure 3 The description is comparable to and lower than Figure 5 The formulation in the mixture was contacted with human plasma for 2 minutes. After subtracting the experimental blank, particularly within this concentration range, mixtures 1 and 2 according to the invention increased the in-situ oxidized quercetin. MoO4 was tested. 2- And La(MoO4) 1- The final concentrations were: 0.00 nM, 3.80 nM, 7.59 nM, 1215 nM and 0.00 nM, 3.25 nM, 6.50 nM, 1040 nM, respectively. Figure 8 ).

[0670] The difference in the amount of quercetin oxide produced between the two formulations (mixture 1 and mixture 2) (apex approximately 295 nm) is not significant. The redox potency of the two compositions according to the invention is comparable.

Claims

1. Use of a topical composition or mixture in the preparation of a medicament for the therapeutic treatment of HSV-1 and / or HSV-2 viral infections, wherein said composition or mixture comprises: Metal salts, wherein the metal is selected from molybdenum (Mo), tungsten (W), vanadium (V), and gold (Au); Chelating agent, selected from BAPTA, EGTA and any mixture thereof; Peroxy free radical source; A buffer that adjusts the pH to 4.0–5.2; The composition or mixture has a redox potential of 300 mV to 450 mV.

2. The use according to claim 1, characterized in that, The composition has a redox potential of 300 mV to 420 mV.

3. The use according to claim 1 or 2, characterized in that, The metal salt is in the form of a peroxide metal salt or a hydrogenated peroxide metal salt, and the metal is in its highest valence state.

4. The use according to claim 1, characterized in that, The metal is oxidized to the highest degree of oxidation.

5. The use according to claim 3, characterized in that, The metal salt is a molybdenum salt, or the metal salt contains molybdenum.

6. The use according to claim 1, characterized in that, The metal salt further includes lanthanum salt.

7. The use according to claim 6, wherein the metal salt is a mixture of molybdenum salt and lanthanum salt.

8. The use according to claim 5, characterized in that, The molybdenum salt has a peroxide unit or a hydrogenated peroxide unit.

9. The use according to claim 1, characterized in that, The metal salt is MoO4. 2- Type or MoO4 2- The hydrogenated-peroxy form.

10. The use according to claim 1, characterized in that, The composition or mixture contains at least two chelating agents.

11. The use according to claim 1, characterized in that, The chelating agent has at least three coordinating groups at the three ends of the molecule forming the chelating agent, the coordinating groups being separated by a chain of at least three atoms, the at least three atoms including at least a nitrogen atom.

12. The use according to claim 1, characterized in that, The chelating agent has at least three carboxylic acid groups.

13. The use according to claim 1, characterized in that, The concentration of the main peroxy free radical source is 200mM~600mM.

14. The use according to claim 1, characterized in that, The concentration of the main peroxy free radical source is 300mM~500mM.

15. The use according to claim 1, characterized in that, The concentration of the main peroxy free radical source is 340mM~450mM.

16. The use according to claim 1, characterized in that, The mixture contains a buffer to maintain a pH of 4.4 to 5.

0.

17. The use according to claim 1, characterized in that, The mixture contains carboxylic acid as a buffer.

18. The use according to claim 1, characterized in that, The mixture contains acetic acid as a buffer.

19. The use according to claim 1, characterized in that, The mixture has a redox potential of 350 mV to 420 mV.

20. The use according to claim 8, wherein, Peroxymolybdate or hydrogenated peroxymolybdate includes (Mo2O6). 4+ or [Mo4O] 12 (O2)2] 4+ .

21. The use according to claim 3, wherein, The amount and ratio of the metal salt and the chelating agent present are sufficient to produce a {peroxide-metal acid salt-chelating agent} complex.

22. The use according to claim 1, wherein, The mixture or composition is prepared by the following method, the method comprising: (i) preparing a buffer solution (BS) containing a buffer of acidic pH; (ii) preparing a solution (SC) containing a metal complex of a metal oxide salt; (iii) preparing a first initial solution (Si1) containing hydrogen peroxide; (iv) preparing a second initial solution (Si2) by mixing the BS solution with the Si1 solution; (v) preparing a solution (S1) containing a peroxide metal compound by mixing the SC solution with the Si2 solution; (vi) preparing a S2 solution by adjusting the pH of the S1 solution with an alkali, the pH of the S2 solution being more alkaline than the pH of the BS solution; (vii) adding one or more chelating agents selected from BAPTA, EGTA, and any mixtures thereof to the S2 solution; and obtaining the mixture or composition.

23. The use according to claim 22, characterized in that, The method includes (viii) adjusting the pH.

24. The use according to claim 22, characterized in that, The method includes (ix) adjusting the volume of the final solution.

25. The use according to claim 22, characterized in that, In the Si1 solution, the concentration of hydrogen peroxide is 200 mM to 600 mM.

26. The use according to claim 22, characterized in that, In the Si1 solution, the concentration of hydrogen peroxide is 300 mM to 500 mM.

27. The use according to claim 22, characterized in that, In the Si1 solution, the concentration of hydrogen peroxide is 330 mM to 460 mM.

28. The use according to claim 22, characterized in that, The buffer in the BS solution is a carboxylic acid / carboxylate buffer, and the ratio of hydrogen peroxide to carboxylate in the BS solution is 20 / 1 to 1 / 1.

29. The use according to claim 22, characterized in that, The buffer in the BS solution is a carboxylic acid / carboxylate buffer, and the ratio of hydrogen peroxide to carboxylate in the BS solution is approximately 5 / 1.

30. The use according to claim 22, characterized in that, The method includes measuring the redox potential in steps (iii) to (ix).

31. The use according to claim 22, characterized in that, The peroxide metal compound in the S1 solution is a peroxide molybdenum compound.

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