PREVENTIVE AND CURATIVE PEROXOMETALLATE BASED COMPOSITION, NOTABLY PHARMACEUTICAL COMPOSITION

Abstract

The invention concerns a mixture or a composition, that is preferably therapeutically active by topical administration, comprising:at least one metal salt, the metal being chosen from molybdenum (Mo), tungsten (W), vanadium (V), gold (Au), a lanthanide, in particular lanthanum; at least one chelating agent; at least one source of peroxidative radicals; at least one buffer agent; and pharmaceutical compositions constituted by or comprising said mixture, the methods for producing same and applications thereof, in particular in a method for the therapeutic treatment of a viral infection, and in particular involving a virus of the Herpesviridae family; or as an anti-inflammatory.

Claims

1. A method of preventing a viral infection involving HSV-2, wherein said method comprises administering to a subject in need thereof an effective amount of a topical pharmaceutical composition, wherein said composition comprises a therapeutically active mixture comprising: at least one metal salt, the metal being selected from the group consisting of molybdenum (Mo), tungsten (W), vanadium (V), gold (Au), and lanthanide; at least one buffering agent; at least one chelating agent; at least one source of peroxidizing radicals; and at least one buffering agent.

2. A method for a therapeutic treatment of genital herpes involving HSV-2, wherein said method comprises administering to a subject in need thereof an effective amount of a topical pharmaceutical composition, wherein said composition comprises a therapeutically active mixture comprising: at least one metal salt, the metal being selected from the group consisting of molybdenum (Mo), tungsten (W), vanadium (V), gold (Au), and lanthanide; at least one buffering agent; at least one chelating agent; at least one source of peroxidizing radicals; and at least one buffering agent.

3. A method for a therapeutic treatment of a viral infection involving HSV-2, wherein said method comprises administering to a subject in need thereof an effective amount of a topical pharmaceutical composition, wherein said composition comprises a therapeutically active mixture comprising: at least one metal salt, the metal being selected from the group consisting of molybdenum (Mo), tungsten (W), vanadium (V), gold (Au), and lanthanide; at least one buffering agent; at least one chelating agent chosen from BAPTA, EGTA and their mixtures; at least one source of peroxidizing radicals; and at least one buffering agent.

4. The method according to claim 1, wherein the method is performed for preventive treatment of an infection involving orofacial-labial, vaginal-anal and/or genital herpes.

5. The method according to claim 1, wherein the method is performed for preventive treatment of orofacial-labial herpes.

6. The method according to claim 1, wherein the method is performed for preventive treatment of genital herpes.

7. The method according to claim 1, wherein the method is performed for preventive treatment of vaginal-anal herpes.

8. The method according to claim 1, wherein the topical pharmaceutical composition, comprises or consists of a therapeutically active mixture comprising: from 0.1 to 100 μM of metal salt of molybdenum; and wherein said composition or mixture has a redox potential from 250 to 550 millivolts.

9. The method according to claim 1, wherein said therapeutically active mixture has a redox potential from 300 to 450 millivolts.

10. The method according to claim 1, wherein said therapeutically active mixture comprises from 1 to 50 μM of metal salt of molybdenum.

11. The method according to claim 1, wherein said therapeutically active mixture comprises from 5 to 30 μM of metal salt of molybdenum.

12. The method according to claim 1, wherein said therapeutically active mixture comprises a peroxo or hydro-peroxomolybdate complex or a mixture thereof.

13. The method according to claim 1, wherein said therapeutically active mixture comprises (Mo.sub.2O.sub.6).sup.4+ or [Mo.sub.4O.sub.12(O.sub.2).sub.2].sup.4+ or a mixture thereof.

14. The method according to claim 1, wherein the chelating agent is selected from among BAPTA, EGTA and any mixtures thereof.

15. The method according to claim 1, wherein said therapeutically active mixture comprises 0.1 to 100 μM of BAPTA.

16. The method according to claim 1, wherein said therapeutically active mixture comprises 5 to 20 μM of BAPTA.

17. The method according to claim 1, wherein said therapeutically active mixture comprises from 20 to 80 μM of BAPTA.

18. The method according to claim 1, wherein said therapeutically active mixture comprises from 0.1 to 1 mM EGTA.

19. The method according to claim 1, wherein said therapeutically active mixture comprises from 50 to 800 μM EGTA.

20. The method according to claim 1, wherein said therapeutically active mixture comprises from 200 to 2,000 μM EGTA.

21. The method according to claim 1, wherein the molybdenum salt and the chelating agents are present according to a ratio from 10/1 to 1/100, expressed in molar concentrations.

22. The method according to claim 1 wherein said therapeutically active mixture comprises hydrogen peroxide at a concentration ranging from 200 to 600 mM.

23. The method according to claim 1, wherein said therapeutically active mixture comprises from 30 mM to 4.4 M of hydrogen peroxide.

24. The method according to claim 1, wherein said composition is buffered at a pH from 4.0 to 5.2.

25. The method according to claim 1, wherein said composition comprises: Molybdate salt; BAPTA; EGTA; H.sub.2O.sub.2; CH.sub.3COOH/CH.sub.3 COONa; E.sub.redox from 300 to 450 mV; and pH 4.4-5.0.

26. The method according to claim 1, wherein said composition comprises: Molybdate salt; Lanthanum salt, and for example Lanthanum nitrate; BAPTA; EGTA; H.sub.2O.sub.2; CH.sub.3COOH/CH.sub.3COONa; E.sub.redox from 300 to 450 mV; and pH 4.4-5.0.

27. The method according to claim 1, wherein said composition comprises from 0.001 to 5 mM of said pharmaceutically active mixture.

28. The method according to claim 1, wherein the administering of the composition is on a mucosa of the subject.

29. The method according to claim 3, wherein the administering of the composition is on a mucosa of the subject.

30. The method according to claim 1, wherein the patient suffers from herpetic keratitis.

31. The method according to claim 3, wherein the patient suffers from herpetic keratitis.

Description

[0254] In the figures:

[0255] FIG. 1 represents the variations of the redox potential and of the H.sub.2O.sub.2 concentration during the preparation of the Mixture 1.

[0256] FIG. 2 illustrates an Fe.sup.3+ abacus used for assaying the redox efficiency of the Mixture 1.

[0257] FIG. 3 is an illustration of the redox efficiency of the Mixture 1 at different concentrations thereof (absorbance of quercetin by Fe(III) in a human plasma medium after contact (2 mins) with the Mixture 1 (in nM MoO.sub.4.sup.2−).

[0258] FIG. 4 illustrates an abacus of the redox efficiency of the Mixture 1 at different concentrations of the latter.

[0259] FIG. 5 illustrates the influence of the Mixture 1 at different concentration of MoO.sub.4.sup.2− on the production in situ in human plasma of Fe.sup.3+, i) stemming from the transferrin-Fe.sup.3+ complex after contacting with the formulation, ii) on the Fe.sup.2+/Fe.sup.3+ equilibrium and, iii) on the oxidation of quercetin.

[0260] FIG. 6 illustrates the production of interleukin-6 (IL-6) in the culture supernatants of non-pathological human skin biopsies (2 mins) stimulated by the Mixture 1 (24.3 μM of active substance) or NaCl (100 mM) as a control. (D1-5: donor 1 to 5, B1 or 2: biopsy 1 or 2).

[0261] FIG. 7 illustrates the average of the production at 6 h and 24 h after contact with interleukin-6 (IL-6) in the culture supernatants of non-pathological stimulated human skin biopsy (2 mins) by the Mixture 1 (24.3 μM of the active substance) or NaCl (100 mM) as a control.

[0262] FIG. 8 is a representation of the redox effectiveness of Mixture 1 and Mixture 2 at different concentrations (absorbance of quercetin by Fe(III) in human plasma media after contact (2 min.) with Mixture 1 (in nM MoO.sub.4.sup.2−) or with Mixture 2 (in nM La(MoO.sub.4).sup.1−).

EXAMPLES

Example 1—Example of an Active Mixture According to the Invention

[0263] The present example is made with a molybdenum salt. A composition of a formula according to table 1 (“Mixture 1”), expressed in the initial concentration of the constituents is prepared:

TABLE-US-00001 TABLE 1 Mixture according Components to the invention Sodium molybdate 20.7 μM BAPTA 12.6 μM EGTA 526 μM H.sub.2O.sub.2 353 mM CH.sub.3COOH/CH.sub.3COONa 70 mM Ph (with NaOH) 4.4-5.0 E°.sub.redox 300 to 420 Mv

[0264] The composition is said to be an «initial» composition since it corresponds to the concentrations of the added reagents without taking into account the applied catalytic process.

[0265] All the constituents used for the synthesis as well as the finished product are validated by their IR-FT spectrum.

TABLE-US-00002 TABLE 1a Product Purity Raw formula CAS No. Sodium molybdate dihydrate 98-103% Na.sub.2MoO.sub.4•2H.sub.2O 10102-40-6 BAPTA: (1,2-bis(o-aminophenoxy)ethane-  ≥98% C.sub.22H.sub.24N.sub.2O.sub.10 85233-19-8 N,N,N′,N′-tetraacetic acid) EGTA: (ethylene glycol-bis(2-aminoethylether)-  ≥99% C.sub.14H.sub.24N.sub.2O.sub.10 67-42-5 N,N,N′,N′-tetraacetic acid) Hydrogen peroxide  29-31% H.sub.2O.sub.2 722-84-1 Glacial acetic acid 99.8-100.5%    CH.sub.3COOH 64-19-7 Sodium acetate trihydrate 99-101% C.sub.2H.sub.3NaO.sub.2•3H.sub.2O 5010524 Sodium hydroxide 10N NaOH 1310-73-2 Demineralized water 1 μS H.sub.2O 7732-18-5

[0266] The figures of the measurements carried out during and after the synthesis are the averages from the 3 productions.

[0267] The amounts indicated below are for the preparation of one liter of Mixture 1.

[0268] A—Step (i): Preparation of the Buffering Solution (Solution BS)

TABLE-US-00003 CH.sub.3COOH (100%; δ: 1.05 g .Math. cm.sup.−3 4.0 ml (final 70 mM) (liquid, 20 C.)) CH.sub.3COONa•3H.sub.2O (100%) 8 g (final 59 mM) Water qsp 1 L Verification of the pH: 4.7-4.8 Room temperature (20° C.)

[0269] B—Step (ii): Preparation of the Metal Complex Solution (Solution CS)

TABLE-US-00004 Na.sub.2MoO.sub.4•2H.sub.2O (source of Mo(VI); 100%) 200 mg (final 2.4 mM) Water qsp 340 ml Mild stirring, room temperature

[0270] C—Step (iii): Preparation of the Initial Solution (Si1)

TABLE-US-00005 Water 900 mL CH.sub.3COOH (100%) 4 mL (70 mM) Add very slowly and with very mild stirring (250 rpm), pH: 2.9-3.8 E.sub.redox: 470-490 mV and then, H.sub.2O.sub.2 (30%) qsd for final 1.2% by weight and according to the preliminary assays

[0271] At 45 mins:

TABLE-US-00006 pH: 2.70-2.90 E.sub.redox: 440-460 mV By the Noble equation: (Solution Si1) [H.sub.2O.sub.2] = 380-410 mM

[0272] D—Step (iv): Preparation of the Initial Solution (Si2)

TABLE-US-00007 Introduce the « BS solution » into the 12 mL (≈1 mM acetate) solution Si1 Add with mild stirring.

[0273] At 45 mins:

[0274] pH: ≈2.90

[0275] E.sub.redox: 460-480 mV

TABLE-US-00008 By the Noble equation: [H.sub.2O.sub.2] = 360-410 mM (Solution Si2)

[0276] E—Step (v): Preparation of the Peroxomolvbdenum Solution S1

TABLE-US-00009 Introduce the « CS solution » into the solution Si2 10 mL

[0277] At 45 mins:

TABLE-US-00010 pH: 2.80-3.00 E.sub.redox: 450-480 mV By the Noble equation: [H.sub.2O.sub.2] = 370-400 mM (Solution S1)

[0278] F—Step (vi): Preparation of the Solution 2

[0279] Adjustment of the pH with mild stirring:

TABLE-US-00011 NaOH 9.9-10.1M 3.6 mL for = 0.9 L of the mixture 1 pH 4.5-5.0 At 45 mins: pH: 4.60 E.sub.redox: 380-390 mV (Solution S2)

[0280] G—Step (vii): Addition of the Chelating Agents—Preparation of the Solution S4

TABLE-US-00012 Introduction of BAPTA (98.8%) 6 mg/L, after very slow dissolution of BAPTA with mild stirring in an aliquot of the formulation S2 (1.4 L) equilibrated beforehand at 25° C. (the so called « S3.1 » solution).

[0281] Pool of the solution S3.1 with the solution S2 in order to form the solution S3.2.

[0282] At 45 mins:

[0283] pH: 4.40-4.70

[0284] E.sub.redox: 380-390 mV

[0285] By the Noble equation: [H.sub.2O.sub.2]=370-390 mM

TABLE-US-00013 Introduction of EGTA (99.1%) 200 mg/L in the solution S3.2 in order to form the solution S4
the solution S4

[0286] Dissolve with mild stirring.

[0287] At 45 mins:

[0288] pH: 4.40-4.60

[0289] E.sub.redox: 380-390 mV

[0290] By the Noble equation: [H.sub.2O.sub.2]=370-420 mM

[0291] H—Step (viii): Adjustment of the pH

[0292] Adjustment of the pH (NaOH 9.9-10.1M, i.e. ≈400 g/L)=>pH: 4.60±0.2

[0293] I—Step (ix): Adjustment of the Volume for the Final Solution (FS)

TABLE-US-00014 Adjustment of the volume with demineralized water qsp 1 liter

[0294] At 12-18 hours:

[0295] pH: 4.50-4.70

[0296] E.sub.redox: 380-390 mV

[0297] By the Noble equation: [H.sub.2O.sub.2]=350-380 mM

[0298] By titration: [H.sub.2O.sub.2]=400-430 mM

[0299] Density: δ=1.004 g/ml

[0300] The mixture according to the invention, ready to use, is stable in darkness for more than 6 months at room temperature or at 45° C.

[0301] Na hydro-peroxo-molybdate is at a concentration of 24.3 μM in the final Mixture 1.

Example 2—Signature by Variation of the Redox Potential

[0302] The signature of the active mixture according to the present invention is evaluated by the variation of the redox potential during the synthesis of a composition prepared according to Example 1.

[0303] According to FIG. 1, relatively to the initial solution(≈470 mV; step 3), reduction of the redox potential of ≈90 mV (≈280 mV) in step 6 (adjustment of the pH with soda). The redox potential remains constant until the end of the synthesis (step 9). The same applies during the ageing of the formulations of the mixture of the invention according to example 1 over 6 months at room temperature (389 mV±5 mV) and at 45° C. (389 mV±8 mV) for a pH of 4.56±0.40 and 4.53±0.41, respectively.

Example 3—Signature by Consumption and Production of H.SUB.2.O.SUB.2.—Generation of the Peroxo-Reagent Equilibrium and of the Redox Potential of Mixture 1

[0304] The signature of the active mixture according to the present invention is evaluated by consumption and production of H.sub.2O.sub.2 during the synthesis of a composition prepared according to example 1.

[0305] According to FIG. 1, relatively to the H.sub.2O.sub.2 concentration of the initial solution (353 mM; step 3 to 10), a sudden peroxide production (≈42 mM; including a small fraction of peracetic acid) is observed because of the addition of H.sub.2O.sub.2 into the buffering solution (acetic acid/acetate). If the addition of H.sub.2O.sub.2 is not carried out in an aqueous solution acid-buffered beforehand (step 3; pH=2.84±0.1), its spontaneous and rapid degradation would result from this because of a too high pH (water) (pKa.sub.HO2⋅-/O2⋅-: 4.8).

[0306] From step 4 up to step 7, a limited consumption of H.sub.2O.sub.2 is observed (10 mM±6 mM), until its stabilization at 381 mM±5 mM (step 7; +BAPTA).

[0307] The addition of EGTA displaces the peroxide equilibrium (Fenton «like» reaction) by peroxide degradation, i.e. 393 mM±29 mM in step 7, to 357 mM±26 mM in step 9, and 365 mM±15 mM in step 9+12 hours.

[0308] In step 6, a sudden drop in the redox potential is observed (from 463 mV±15 mV to 392 mV±17 mV) by adjustment of the pH is observed. The addition (step 7) of the chelating agents BAPTA and then EGTA successively contributes to stabilization of the redox potential (386 mV±5 mV and 383 mV±6 mV) by stabilization of the chemical equilibria.

Example 4—Assay of the Redox Efficiency of the Mixture of the Invention: Quercetin Methods

[0309] A—The Reactive Solutions

[0310] 1—Quercetin

[0311] A solution 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.sub.15H.sub.10O.sub.7.2H.sub.2O) at 10.sup.−3 mol/L is prepared in methanol.

[0312] 2—Hydrochloric Solution

[0313] A first solution of hydrochloric acid 1M is prepared in methanol. A second solution is prepared at 0.3 M in methanol. The reactive solution is prepared by adding 343 μl of the solution 1M to 1,357 μl of the 0.3 M solution.

[0314] 3—Solutions of Fe.sup.3+ (Reference Abacus)

[0315] A solution of Fe.sup.3+ (Fe.sub.2(SO.sub.4).sub.3.xH.sub.2O) at 2 mg/ml is prepared in HPLC water. It is then diluted in order to obtain eight reference solutions at: 20, 100, 200, 300, 400, 500, 1,000 and 1,500 μg/ml. These solutions are finally diluted 20 times in human plasma or in HPLC water in order to finally obtain the concentrations of: 0, 1, 5, 10, 15, 20, 25, 50 and 75 μg/ml.

[0316] 4—Solutions of Fe.sup.2+ (Reference Abacus, Activity Control)

[0317] A solution of Fe.sup.2+ (FeSO.sub.4.7H.sub.2O) at 89 mg/ml is prepared in HPLC water. It is then diluted in order to obtain three reference solutions at: 0.445, 4.45 and 44.5 mg/ml. These solutions are finally diluted 10 times in HPLC water in order to finally obtain concentrations of: 0, 0.0445, 0.445 and 4.45 mg/ml.

[0318] B—Assays with the Quercetin Method

[0319] The presence of Fe.sup.3+ (abacus) or its production (Fe.sup.2+ abacus and efficiency of the mixture of the invention after 2 mins of contact) are quantified by the quercetin method.

[0320] According to the conditions of analysis or use of the Mixture 1:

[0321] “mixture.sub.ox” refers to the mixture according to the invention as an oxidizer.

[0322] “mixture.sub.red” refers to the mixture according to the invention as a reducing agent.

[0323] The Fe.sup.3+ productions are the following:

Transferrin-2Fe.sup.3++mixture.sub.red+3H.sup.+.fwdarw.Transferrin.sub.red+2Fe.sup.3+.sub.(aq)+mixture.sub.ox   (1)

Fe.sup.3+.sub.(aq)+mixture.sub.red+e.sup.−⇄Fe.sup.2+.sub.(aq)+mixture.sub.ox   (2)

Fe.sup.3+.sub.(aq)+quercetin.sub.red.fwdarw.quercetin.sub.ox+Fe.sup.2+.sub.(aq)   (3) [0324] The reaction (1) is in a plasma medium. [0325] The reaction (2) is in a plasma medium or in HPLC water and corresponds to the equilibrium of the Fenton-Haber-Weiss reaction. [0326] The reaction (3) is the oxidation reaction of quercetin for evaluating the redox potential of the Mixture 1.

[0327] In an acid medium and at 70° C., quercetin is specifically oxidized by ferric iron (Fe.sup.3+; El Hajji et al 2006 and Balcerzak et al, 2008) borne by the transferrin in a plasma medium and not by ferrous iron (Fe.sup.2+). The Fe.sup.3+ ion is soluble under these conditions.

[0328] 30 μl of the quercetin solution are added to 170 μl of the hydrochloric reactive solution. After homogeneization, 50 μl of the sample (Fe.sup.3+ in human plasma or in HPLC water, Fe.sup.2+ in HPLC water, the mixture of the invention after 2 mins of contact with human plasma) are added. The solutions are intensively stirred and shortly and then incubated for 1 h at 70° C. After incubation, the samples are centrifuged for 15 min. at 14,000 rpm and at room temperature. A 100 μl of supernatants are sampled. The UV spectrum of each sample is acquired from 230 to 500 nm. The blanks: water, Fe.sup.2+, Fe.sup.3+, plasma are subtracted according to the type of experiment. The absorption peak of oxidized quercetin by the produced Fe.sup.3+ from plasma transferrin and the Fenton-Haber-Weiss reaction in situ is retained (from 285-305 nm).

[0329] The apex of the curve is centered towards 292 nm when the experiment is carried out in HPLC water or in plasma. It may be moved by a maximum of 10 nm when the experimental blanks are subtracted.

[0330] 1—Fe.sup.3+ Abacus

[0331] An abacus for the Fe(III) ion is produced for example by measuring the UV absorbance (on the range 250-330 nm) of oxidized quercetin for different concentrations of Fe.sup.3+ (cf. the Fe.sup.3+ range above) in human plasma, and by reporting the optical density versus the wavelength.

[0332] From the spectrum of the region 250-330 nm, the area under the curve (AUC) between 285 and 305 nm is calculated with the formula:

[00001]${\mathrm{AUC}}_{285nm}^{305nm}={\int }_{285nm}^{305nm}f\left(x\right)\mathrm{dx}={\int }_{285nm}^{305nm}\mathrm{Absorbance}.d\left(\lambda \right)$

Accordingly, each area of the surface of the peak 285-305 nm for each point of the range of Fe.sup.3+ is plotted for graphic illustration. The tendency curve is plotted (Excel), the equation (the correlation coefficient R.sup.2 for which the closest value to 1 validates the experiment) is deducted (Excel).

[0333] It may be a linear or polynomial form of the type: y=ax.sup.2+bx+c. By experiment, this relationship may be of the type

y=−0.0021x.sup.2+0.4819x+0.4521; R.sup.2=0.9966 (FIG. 2).   (4)

[0334] For a mixture according to the invention, the concentration of Fe.sup.3+ equivalent produced in a plasma medium or from Fe.sup.2+ is the reflection of the oxidizing potential of the invention and is calculated from equation (4). This is an indirect method for measuring the redox efficiency of the Mixture 1 by assaying in UV light the oxidized quercetin by Fe.sup.3+. Accordingly, a concentration in Fe.sup.3+ «equivalent» is mentioned. Also, it is possible to compare the redox potential of a solution according to the invention to be tested, relatively to a reference solution according to the invention, in other words making a comparison with the quantification of the redox efficiency of a mixture according to the invention.

[0335] 2—Fe.sup.2+ Abacus

[0336] In the same way as for the ion Fe(III), an abacus is produced for the Fe(II) ion is produced. For example, the UV absorbance (230-500 nm) of the quercetin peak oxidized by the Fe.sup.3+ obtained from the mixture of the invention at 1.22 μM of active substance (250 μg/L) and 2 mins of contact with Fe.sup.2+ is for example measured for the range described above. The same mathematical analyses of AUC as described for the Fe(III) ion may be conducted.

[0337] 3—Assay of the Redox Efficiency of the Mixture of the Invention for Example at 1.22 μM (250 μg/L) of Active Substance MoO.sub.4.sup.2−.

[0338] According to the procedure described in B—, the redox efficiency of a mixture according to the invention (1.22 μM of MoO.sub.4.sup.2− in human plasma) is tested for: i) evaluating its ageing at room temperature and at 45° C., ii) validating its production and comparative evaluating the formulations, iii) quantifying its transcutaneous passage, iv) its bioavailability and, v) its biophase.

[0339] According to the equation (4), the specific area of the UV absorbance peak is calculated (as an AUC) between 285 and 305 nm of the quercetin peak oxidized by the free Fe.sup.3+ produced after plasma contact for 2 mins of the mixture of the invention (equations (1), (2) and (3)) at a final concentration finale of 1.22 μM with the transferrin-Fe.sup.3+ complex. On average, the production of Fe.sup.3+ equivalent (Fe.sup.3+.sub.eq) is of the order of 30 to 50 μg/ml of plasma.

[0340] 4—Estimation Abacus in a Plasma Medium of the Effective Concentration of the Mixture According to the Invention

[0341] Plasma iron is not free. It is bound to transferrin (or siderophilin) in its ferric form (Fe.sup.3+) in amount of 1 to 2 residues per molecule. Iron in its ferrous form (Fe.sup.2+) is not circulating outside its complexation with hemoglobin. It is considered that the human plasma used is not hemolyzed. The quercetin reaction of the inventive mixture with plasma copper is considered as negligible.

[0342] A range of concentrations of the mixture of the invention (15.2, 7.6, 3.8 and 1.9 nM of final MoO.sub.4.sup.2−) was incubated in human plasma. The reaction with quercetin was conducted as described in B—. The specific absorption peak (substraction of the plasma blank +quercetin) was integrated as described in B-1 (FIG. 3).

[0343] The abacus was plotted (Excel) in a polynomial scale and the tendency equation was calculated (y=0.0144x.sup.2+0.5863x+0.4511 with R.sup.2=0.9934 (5); FIG. 4).

[0344] A proportionality relationship (equation 5) is ascertained between the amount of the invention in an added concentration to human plasma and the amount of equivalent of Fe.sup.3+ (equation 4) detected by oxidation reaction of quercetin, i.e. 28.95, 12.19, 3.97, 3.12 μg of Fe.sup.3+.sub.eq/mL of human plasma, according to the range of the mixture of the invention described above.

Example 5—Evaluation of the Oxidation-Reduction Efficiency of the Invention on Human Plasma

[0345] After adding the Mixture according to the invention (Exampl 1—Mixture 1”) to human plasma and after a contact time of 2 mins, up to a concentration of 0.03038 μM of MoO.sub.4.sup.2−, the oxidation reaction of quercetin for the production of Fe.sup.3+ issued from the plasma transferrin-Fe.sup.3+ complex and the Fenton-Haber-Weiss reaction is optimum and linear (cf. example 4).

[0346] After substraction of the experimental blanks, an increase in the concentration of the invention generates a reduction in the production in situ of quercetin oxidized by Fe.sup.3+ because of the displacement of the equilibria of the Fenton-Haber-Weiss reaction (Fe.sup.3+⇄Fe.sup.2+ ). The final tested concentrations of MoO.sub.4.sup.2− were then 0.00, 1.22, 4.05, 12.15, 24.30 and 30.38 μM (FIG. 5).

Example 6—Anti-Replication Efficiency

[0347] A—The Models

[0348] Four contact models of the mixture according to example 1 (“Mixture 1”) were tested. They represent the four physiological possibilities that the Mixture 1 may be encountered during a topical therapeutic application, i.e.:

[0349] Model 1: Mixture 1 on cells infected with HSV-1,

[0350] Model 2: Mixture 1 on cells not yet infected with HSV-1,

[0351] Model 3: Mixture 1 on cells not yet infected and Mixture 1 on HSV-1 free (not yet infectious),

[0352] Model 4: Mixture 1 on HSV-1 before infection.

[0353] Two contact times of the Mixture 1 were tested, i.e. 2 mins or 1.5 mins, and then removal of the Mixture 1.

[0354] Two concentrations of the Mixture 1 were tested: 0.81 and 2.03 μM of active substance, i.e. 167 and 417 μg/L, respectively.

[0355] The method for quantifying the efficiencies 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.)

[0356] The anti-replication efficiency (AE) of the Mixture 1 on HSV 1 was evaluated in qPCR with specific primers of the viral genome (Kessler H. H. et al (2000) “Detection of Herpes simplex virus DNA by real-time PCR” J. Clin. Microbiol., 38(7), 2638-2642).

[0357] The cytotoxicity of the Mixture 1 (C) on the BHK-21 line was evaluated in qPCR with specific primers of the gene of the small ribosomal sub-unit 18S (Texcell—Evry, France).

[0358] The multiplicity of infection (MOI): 1, i.e. 5.10.sup.5 TCID.sub.50 (Tissue Culture Infective Dose 50%).

[0359] The qPCR experiments were conducted at t0 h (2 h after contact between BHK-21 and HSV-1, and then removal of HSV-1), t2 h- , t4 h- and t8 h-post-infection.

[0360] The AE/C ratio gives us the Efficiency Index in vitro (EI) of the Mixture 1 corresponding to a study model.

[0361] Model 1:

[0362] BHK-21+HSV-1 (2 h; infection)=>washing (PBS) for viral removal=>samples t0 h=>+Mixture 1 (2 min.)=>washing (culture medium) for removing Mixture 1=>cell incubation at 37° C.=>samples t2 h-, t4 h- and t8 h-post-infection.

[0363] Model 2:

[0364] BHK-21+Mixture 1 (1.5 and 2 mins)=>washing (PBS) for removing the Mixture 1 =>cells+HSV-1 (2 h; infection)=>washing (culture medium) for removing HSV-1=>samples t0 h=>cell incubation at 37° C.=>samples t2 h-, t4 h- and t8 h-post-infection.

[0365] Model 3:

[0366] BHK-21+Mixture 1 (1.5 and 2 mins)=>washing (PBS) for removing Mixture 1.

[0367] HSV-1+Mixture 1 (1.5 and 2 mins)=>washing (PBS) for removing Mixture 1.

[0368] Pool of the cells and of the viruses having been in preliminary contact with Mixture 1 (2 h; infection)=>washing (culture medium) for removing HSV-1=>samples t0 h=>cell incubation at 37° C.=>samples t2 h-, t4 h- and t8 h-post-infection.

[0369] Model 4:

[0370] HSV-1+Mixture 1 (2 mins)=>washing (PBS) for removing the Mixture 1=>+BHK-21 (2 h; infection)=>washing (culture medium) for removing HSV-1=>samples t0 h=>cell incubation at 37° C.=>samples t2 h-, t4 h- and t8 h-post-infection.

[0371] B—Results

TABLE-US-00015 TABLE 2 Reduction to 8 hours of the multiplication of HSV-1 in BHK-21 cells after contact with Mixture 1 (in μg/L of active substance) or of acyclovir (ACV); Efficiency Index in vitro (EI). Reduction of the HSV-1 replication at t8 h (%)* EI at t8 h/model** Substance Mixture 1 ACV Mixture 1 Mixture 1 ACV Mixture 1 Contact time 2 2 2 1.5 1.5 2 2 2 1.5 1.5 (min.) Concentration 167 417   10.sup.3 167 417 167 417  10.sup.3 167 417 (μg/L) Model 1 −97 −98 −12 NT NT 37 48 2 NT NT Model 2 −72 −96 −1 −70 −86 4 23 1 3 7 Model 3 −93 −97 −6 −78 −90 14 35 1 4 10 Model 4 −56 −69  −52.sup.# NT NT 2 3  2.sup.# NT NT *The reduction of the HSV replication (RR) by the tested substances (Mixture 1, control acyclovir) is expressed as a percentage and corresponds to the qPCR quantifications ratio of the HSV-1 genomes and of the cytotoxicity which is evaluated by quantifying the sub-unit 18S of the host cell: RR = {(100 × ([HSV-1].sub.test/[18S].sub.test))/([HSV-1].sub.contol+/[18S].sub.control+)} − 100. **The anti-replication Efficiency Index in vitro (EI) of the tested substances is represented by the ([HSV-1]/[18S] evaluated in specific qPCR) of the positive control infected by HSV ([HSV-1].sub.control+/[18S].sub.control+) vs. that of the tested substances (Mixture 1, control acyclovir): EI = ([HSV-1].sub.control+/[18S].sub.control+)/([HSV-1.sub.test/[18S].sub.test). #[ACV]: 1 g/L

[0372] The best EIs (Table 2) are obtained after 2 minutes of contact of the mixture 1 and of a concentration at 417 μg/L. We note a significant influence of the contact time on the EIs (1.5 mins and 2 mins; models 2 and 3).

[0373] The EIs obtained with the models 1 and 3 with a contact time of 2 mins and a concentration of the mixture 1 of 167 μg/L, are particularly significant.

[0374] Regardless of the model, the EIs obtained with acyclovir (“ACV”) are not significant (Table 2).

TABLE-US-00016 TABLE 3 Quantification percentages by qPCR (gene 18S or HSV; at 8 h post-contact of 2 mins): i) of the multiplication of the BHK-21 line (control: non-infected line) and, ii) of the replication of HSV-1 (control: infected line). Gene/genome 18S (%) HSV (%) Control Line + (PBS − HSV) Line + (PBS + HSV) Test substance Mixture 1 Mixture 1 Mixture 1 [active 167 417 ACV 167 417 ACV 167 417 ACV substance] μg/L μg/L (mg/L) μg/L μg/L (mg/L) μg/L μg/L (mg/L) Model 1 45 27 108 (1) 49 30 118 (1) 1 <1 76 (1) Model 2 62 41  57 (1) 68 44  62 (1) 19 2 61 (1) Model 3 118 70 111 (1) 98 58  92 (1) 7 2 86 (1) Model 4 120 107 97 139 123 111 64 38 54 Tests 1 2 3 4 5 6 7 8 9 indicates data missing or illegible when filed

[0375] For the Mixture 1 (at 167 to 417 μg/L) or ACV (at 1 or 10.sup.3 mg/L), regardless of the control used (line+PBS−HSV or line+PBS+HSV), the quantification results of 18S are comparable for a same model (Table 3, test 1 vs. 4, 2 vs. 5, 3 vs. 6).

[0376] For the Mixture 1, a dose-dependence remarkable anti-replication efficiency (Table 3, tests 7 and 8) is ascertained for the models 1, 2 and 3. This is not observed for ACV (Table 3, test 9).

[0377] A comparable cytotoxicity of the Mixture 1 (167 μg/L) and of ACV (1 mg/L) (Table 3) is noted according to the models 2 and 3 (Table 3, tests 1, 3, 4 and 6).

[0378] An absence of anti-HSV efficiency (Table 2 and Table 3, test 9) for ACV (1 mg/L or 1g/L) is noted. The models introduced for the study of the Mixture 1 cannot correspond to a study dealing with ACV.

[0379] For model 1 (Mixture 1 on infected cells):

[0380] The best EI (Table 2) with two concentrations of the mixture 1 (2 mins of contact, 167 and 417 μg/L, El: 37 and 48, respectively).

[0381] The Mixture 1 has direct efficiency (Tables 2 and 3) on intranuclear viral replication, on cell metabolism probably.

[0382] The Mixture 1 has good specificity of the infected cell (Table 3, tests 4 and 5; model 1 vs. 2).

[0383] For the Model 2 (Mixture 1 on a Cell Before Infection): p An interesting EI (Table 2; contact for 2 mins and 417 μg/L, EI: 23).

[0384] The Mixture 1 has an action on the cell which becomes less receptive to the infection and/or acquires a metabolism incompatible with the replication of the virus (Table 3, tests 7 and 8).

[0385] For the Model 3 (Mixture 1 on: Non-Infected Cell and HSV-1, Separately and Before the Pool):

[0386] An interesting EI (Table 2) with two concentrations of the mixture 1 (2 mins of contact, 167 and 417 μg/L, EI: 14 and 35, respectively).

[0387] Depending on the applied dose (167 or 417 μg/L) zero cytotoxicity (Table 3, test 4) or limited cytotoxicity (Table 3, test 5) for very good antiviral efficiency (Table 3, tests 7 and 8, respectively).

[0388] We have an efficiency of the mixture 1 which would be an accumulation of the model 2 with a significant action on the cell and of the model 4 with a limited action on the virus alone (Tables 2 and 3).

[0389] For the model 4 (Mixture 1 on HSV-1):

[0390] A low EI (contact time: 2 mins, 167 and 417 μg/L, EI: 2 and 3, respectively; Table 2) which is confirmed by absence of cytotoxicity but significant viral replication (Table 3, tests 8 and 9).

[0391] C—Conclusions

[0392] On the BHK-21 line, the main efficiencies of the Mixture 1 is: i) of having a preferential cytotoxicity of the infected cell, ii) of inhibiting the viral replication in the infected cell and, iii) limiting the infection of the healthy cell.

[0393] The target may be a membrane (receptors for the virus, for example) and/or metabolic (regulation of the stress and apoptotic routes, for example).

[0394] Viral Recognition and Internalization

[0395] According to model 4, the Mixture 1 has a low direct anti-replication efficiency at the virus. Under these experimental conditions, close to the topical therapeutical use, this observation is in favor of a limited alteration of the viral receptors to the cell: gB (HSV-2), gC (HSV-1), gD and gH/gL (HSV-1 and -2). The Mixture 1 would only alter in a limited way the viral glycoproteins by its in vitro Fenton-Haber-Weiss reaction.

[0396] Similarly, we do not note the significant influence of the Mixture 1 on the phospholipid membrane of HSV which may perturb the membrane fusion and penetration.

[0397] Under these experimental condition ns, Mixture 1 is not a virucide.

[0398] Viral Replication and Cell Metabolism:

[0399] The qPCR technique uses the cell lyzate at 8 h post-infection. A reduction in the viral replication may result from poor recognition and/or internalization and/or replication (modification of the cell metabolism induced by the infection and/or the Mixture 1).

[0400] If the viral desalting route was altered by the Mixture 1 without altering the other routes and regardless of the model, the resulting EIs would be much smaller because of the cytoplasm accumulation of virions.

[0401] According to model 2 (EI: 23 with 2 mins of contact time and 417 μg/L), low viral replication after preliminary cell contact with the Mixture 1 (Table 2 and Table 3, test 8) is observed.

[0402] According to this model, the BHK-21 cell was in contact at 2 mins with Mixture 1. The contact time with HSV in a complete and nutritive culture medium was 2 hours. The Mixture 1 induced cell modifications having a lifetime lasting for more than 2 hours and further predicting prophylactic efficiency of Mixture 1.

[0403] In addition to the recognition of HSV-1 by the cell being partly affected by the Mixture 1 because of its probable and limited action on the viral glycoprotein gD (38% of replication; Table 2), this recognition may also be affected by an action on the cell receptors to gD (HVEM and nectin) as well as on the two other surface cell receptors to gB and gH/gL which are heparane sulfates and integrins.

[0404] Because of radical permeation (modification of the intracellular or transmembrane redox potential, regulation of the oxidative stress routes), the cell calcium influxes may be moved. These cell calcium influxes are also induced by the HSV infection. However the latter may be demobilized immediately because of the introduction into the Mixture 1 of free and non-permeating chelating agents (limitation of the cell cytotoxicity). Other major cell modifications on the other hand should not be excluded and may correspond to protein oxidations or reductions (cysteinyl bridges, for example) and in particular of structural proteins which may occur in viral endocytosis.

[0405] According to model 3 (Table 2 and Table 3, tests 7 and 8), a significant reduction in the viral replication is observed (EI: 14 and 35 for 2mins of contact time, 167 and 417 μg/L, respectively).

[0406] Both phenomena accumulate. The first is a limited action at the virus (cf. model 4), the second more significant (cf. model 2) is at the cell.

[0407] The addition of the EIs of models 2 and 4 (2 mins and 417 μg/L), i.e. 23 and 3, respectively, gives a sum (26) less than but close to the experimentally obtained one for model 3, i.e. 35.

[0408] A potentialized accumulation i) of poor recognition of the HSV partners and cell is contemplated and ii) an inhibited internalization and/or replication notably by alteration of the cell metabolism.

[0409] Model 1 (EI: 48 with 2 mins of contact time and 417 μg/L) is the most performing.

[0410] The action of the anti-infectious Mixture 1 is preferential on the cell than on the virus. This action is all the more performing since the cell is infected beforehand.

[0411] In this model which corresponds to the best therapeutic problem, it is obvious that the Mixture 1 has a specific major intracellular pharmacological efficiency. For 417 μg/L of active substance, the table 3 shows that the amount of ribosomal sub-units 18S is less (depending on the control used, 27 and 30%, tests 2 and 5) in this model than in the model 2 (depending on the control used, 41 and 44%, tests 2 and 5) and in the model 3 (depending on the control used, 70 and 58%, tests 2 and 5) which is an argument for the specificity of the infected cell vs. the healthy cell.

[0412] The targets of the Mixture 1 in this model cannot be either the recognition or the internalization, but actually metabolic modifications. The latter are placed in a new equilibrium, notably because of the transmembrane radical passage, the modification of the redox potential of the cell and the presence of outer chelating agents, between the oxidative stress routes (already induced by the infection), the calcium fluxes (already induced by the infection), the inhibition of the calcium-dependent release of the virions, the modifications by oxidation of proteins directly or indirectly involved in viral replication.

Example 7—Evaluation Ex Vivo of the Production of Interleukins

[0413] Non-pathological biopsies (8 biopsies and 4 donors) of human skin issued from surgical operations for reducing weight were treated ex vivo with the Mixture 1 (5 mg/L of active substance; 2 mins). The supernatants in which the biopsies were found were sampled at 6 h and 24 h post-contact in order to quantify the interferons-α (IFN-α; inhibition of the infection with HSV; Mikloska Z. et al (2001) “Alpha and Gamma Interferons Inhibit Herpes Simplex Virus Type 1 Infection and Spread in Epidermal Cells after Axonal Transmission”, J. Virol., 75(23), 11821-11826), -β(IFN-β; inhibition of 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; inflammation marker).

[0414] The positive experimental control is a physiological solute (NaCl 100 mM).

[0415] No modification of the cell base level of IFN-α and IFN-β was noted.

[0416] Depending on the biopsies and on the donors and accordingly on the trauma of the surgical operation and of the experimental manipulation:

[0417] At 6 h post-contact (FIGS. 6 and 7):

[0418] with NaCl as a control, production of basal IL-6 of 277 pg/mL±11 to 2,866 pg/mL±206.

[0419] with Mixture 1, inhibition of the IL-6 production relatively to the NaCl control, from 0 pg/mL to 1,015 pg/mL±17.

[0420] At 24 h Post-Contact (FIGS. 6 and 7):

[0421] with NaCl control, basal IL-6 production of 1,485 pg/mL±37 to 7,454 pg/mL±199.

[0422] with Mixture 1, inhibition of the IL-6 production relatively to the NaCl control, of 84 pg/mL±15 to 5,910 pg/mL±29.

[0423] Conclusions:

[0424] production and overproduction heterogeneity (6 h and 24 h, respectively) for IL-6 post-contact NaCl and Mixture 1 (FIG. 6).

[0425] a heterogeneous inhibition of production of IL-6 post-contact with Mixture 1 (5 mg/L of active substance) which is on average 28% at 6 h and 19% at 24 h, relatively to the NaCl control (FIG. 6).

[0426] By this production inhibition, Mixture 1 has the property of being an anti-inflammatory (FIG. 7).

Example 8—Plasma Degradation and ½ Life

[0427] One of the donors of radicals (mainly HO.sub.2.sup.⋅, which has a lifetime up to a few seconds and a notable cell penetration index) by a reaction of the Fenton-Haber-Weiss type of the active substance MoO.sub.4.sup.2− of the Mixture 1 is H.sub.2O.sub.2. The degradation of this tracer was followed for evaluating the consumption rate of the Mixture 1 or its deactivation.

[0428] Technique:

[0429] The Mixture 1 (68.9 μM or final 14.2 μg/L) was incubated in fresh human plasma. The H.sub.2O.sub.2 disappearance kinetics were followed by a peroxidase reaction on aliquots sampled at successive times.

[0430] Results:

[0431] On average (n=10), the percentage of degradation of the tracer substance of the Mixture 1 in human plasma at room temperature after a contact time of 2 mins is of 85.3±9.4%, 74%±15.2% of which in the first minute.

[0432] In the first minute, the “Degradation Time 50%” or DT.sub.50=0.812 mins for a solution of Mixture 1 at an initial 68.9 μM of active substance and initial 1 mM of H.sub.2O.sub.2.

[0433] The degradation rate K.sub.cH2O2: ≈740 μmoles of Mixture 1 at an initial 1 mM of H.sub.2O.sub.2/min./L of plasma or K.sub.cMoO42−: ≈51.0 μmoles of Mixture 1 at initial 68.9 μM of active substance MoO.sub.4.sup.2−/min/L of plasma, in the first minute of incubation at room temperature.

[0434] For a sample of 200 μL of the Mixture 1 at 24.3 μM (5.0 mg/L) of active substance or 353 mM (12 g/L) of the H.sub.2O.sub.2 tracer, within the scope of a topical therapeutic application, there are ≈5 nmoles of active substance MoO.sub.4.sup.2− or 70 μmoles of H.sub.2O.sub.2 tracer. This sample, in 1 L of plasma, is degraded within ≈0.1 minute.

Example 9—Limited Hematotoxicity on Peripheral Blood

[0435] The goal of this study is to evaluate the hematotoxicity of the Mixture 1 (24.3 μM or final 5.0 mg/L) after incubation of 2, 3 and 5 minutes in human peripheral blood. The toxicity for which the haematolysis is evaluated following the main blood parameters, i.e. the counts of erythrocytes, leukocytes, the hematocrit, of the platelets, the average globule volume and the hemoglobin level.

[0436] Conclusions:

[0437] The hematological parameters relating to the counts of erythrocytes, leukocytes, the hematocrit, the average globule volume and the hemoglobin level are not modified by an incubation for up to 5 mins with the mixture according to the invention.

[0438] The platelet count is modified by the contact with Mixture 1 from -35% immediately after as soon as its addition, at −69% at 5 minutes.

Example 10—Limited Transcutaneous Passage of the Mixture According to the Invention, Absorption, Biophase and Bioavailability (OECD 428; EMEA, Human Guideline, 2001)

[0439] The Mixture 1 used was five times more concentrated (Mixture 1 ×5) so as to show the smallest passages of substances. The goal of this study was to evaluate (reference t0, t2 min., to t60 min) the passage of the Mixture 1 ×5 as well as that of the tracer H.sub.2O.sub.2 through non-pathological human skin biopsies.

[0440] This procedure is applied to three types of substrate: total biopsy, dissected biopsy in the epidermis on the one hand and in the dermis on the other hand.

[0441] Technique:

[0442] 12 biopsies from 2 donors were tested (measurements in the drop deposited on the flap [30 μL]) and in the medium below the latter [800 μL]) according to both methods (Table 4): i) quercetin which evaluates the redox efficiency of the Mixture 1 ×5 and, ii) the peroxidase which measures the tracer H.sub.2O.sub.2concentration.

[0443] The drop (30 μL) deposited above the biopsies and the epidermal and dermal flaps is 5 times more concentrated in active substance (121.5 μM or 25.02 mg/L) and in tracer (1.77 M or 60 g/L) than the therapeutic formulation (24.3 μM or 5.0 mg/L and 352.8 mM or 12 g/L, respectively). In the 30 μL of the Mixture 1 ×5 deposit, there are 3.65 nmoles or 750 ng of active substance and 53 μmoles or 1.8 mg of tracer.

Results:

[0444]

TABLE-US-00017 TABLE 4 Test Trial Biopsy* Epidermis* Dermis* Drop 1 Redox efficiency −1.4 to− 10.5 −5.7 to −23.9 NT 2 H.sub.2O.sub.2 tracer −16.5 to −57.7 −72.5 to −99.9 −99.6 Medium 3 Redox efficiency +3.7 to +9.0 +0.8 to +80.6 +44.5 to +100 4 H.sub.2O.sub.2 tracer <+0.001** to +0.09 +0.38 to +1.7 +0.16 to +0.46 *in % relatively to t.sub.0 (−: decrease, +: increase) **detection limit.

[0445] A low-to-moderate decrease (test 1) of the redox efficiency of the Mixture 1 is observed in the drop above the flap (from −1.4 to −23.9%).

[0446] A moderate-to-significant decrease (test 2) of the amount of the tracer H.sub.2O.sub.2 of the Mixture 1 is observed in the drop above the flap (from −16.5 to −99.9%).

[0447] A slight-to-significant increase (test 3) of the redox efficiency of the medium under the flap is observed. It depends on the flap and on its type. When this is a complete biopsy, this increase due to the transcutaneous passage is low. On average it is of 6.35% and corresponds to the passage of 232 pmoles (47.6 ng) of active substance of the Mixture 1. When this is the epidermis or the dermis, this increase is highly variable (from 0.8 to 100%). This is most likely due to differences in the histological structures (pores, dermal blood irrigation, epidermal nerve terminations, for example).

[0448] An increase from non-detectable to slight (test 4) of the amount of the tracer H.sub.2O.sub.2 in the medium under the flap is observed regardless of its type (from <+0.001 to 1.7%). When this is a complete biopsy, this increase due to the transcutaneous passage is particularly low (<0.53 to 47.7 nmoles, i.e. <0.018 to 1.62 μg). When this is the epidermis or the dermis, on average, this increase is low (1.04 and 0.31%, i.e. 0.55 μmoles and 0.16 μmoles, or 18.7 μg and 5.6 μg, respectively).

[0449] Conclusions:

[0450] There is tissue independence (absorption, deactivation, permeation) of the two parameters considered as typical of Mixture 1 which are the redox efficiency due to the active substance and the dosage of the peroxide tracer, a donor of radicals.

[0451] By considering total biopsy, the penetration index of the Mixture 1 corresponds to the thickness of the flap, i.e. 1 mm (average thickness of an epidermis) to 2 mm.

[0452] The epidermis contains many nerve terminations which may be the center of axone desalting of herpetic virions in the patient. They are the targets of the Mixture 1 in the same way as the infected epidermal cells or before a potential infection. By considering the succession of both tissues, the biophase of the Mixture 1 is quasi strictly the one of the thickness of the epidermis (1 mm) and is attained in totality for maximum target tissue absorption.

[0453] By considering that the dermis alone is vascularized, at most 80.6% of the redox efficiency of the Mixture 1 may be again found at its contact and 1.7% of the tracer. Under these experimental conditions, the bioavailability of the Mixture 1 ×5 is of 2.94 nmoles or 605 ng of the active substance and 0.9 μmoles or 30.6 μg of peroxide tracer.

[0454] In a genotoxicity study in vitro, with the Ames test, the mixture according to the invention and its metabolites is not mutagenic in the presence of the activator S9 (5/5 strains of Salmonella), it is mutagenic on 1/5 strain in its absence.

[0455] In local application on the skin either stimulated or not, the mixture according to the invention (24.3 μM or 5.0 mg/L of active substance) does not have any associated irritation toxicity.

[0456] In a buccal and vaginal irritation study, the mixture according to the invention (24.3 μM or 5.0 mg/L) is not irritating (score 0/16) and very weakly (score 1/16), respectively.

[0457] In a highly sensitive study of irritation of the chorioallantoic membrane of a hen egg, the Mixture 1 (18.23 and 24.3 μM or 3.75 and 5.0 mg/L) is moderately irritating (Irritation index or IS=6.8±0.4 and 6.3±0/21, respectively).

[0458] In an acute toxicity study by dermal application, the mixture according to the invention at the doses of 25.0 and 31.3 μg/kg of active substance contained in 60 and 75 mg/kg of H.sub.2O.sub.2tracer, respectively, is well tolerated without any symptoms, for a “Maximum Tolerated Dose” (MTD) and a “Maximum Dose Without Any Observable Detrimental Effect” (MDWNODE) or “No Observed Adverse Effect Level” (NOAEL) of 31.3 μg/kg of active substance and 75 mg/kg of tracer.

[0459] In an acute toxicity study with intravenous injection, the mixture according to the invention at the doses of 10.4 and 12.5 pg/kg of active substance contained in 25 and 30 mg/kg of H.sub.2O.sub.2 tracer, respectively, is tolerated without any notable symptoms for a “Maximum Tolerated Dose” (MTD) of 12.5 μg/kg of active substance.

Example 11—Acceptable Toxicity of the Mixture According to the Invention

[0460] I—Mutagenicity In Vitro (Ames Test—OECD 471)

[0461] The goal of this study was to evaluate the mutagenic activity of the active mixture according to the invention (Mixture 1) and of its metabolites (produced by the S9 fraction of a rat liver) on the strains of Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535.

[0462] Conclusions:

[0463] Without any S9 activation system, the active mixture (7.7 μM or 2.43 μM, i.e. 1.58 mg/L or 0.50 mg/L) according to the invention and its metabolites do not have any mutagenic effects on the TA97a, TA98, TA100 and TA1535 lines.

[0464] With an activation system S9, the active mixture (Mixture 1 ×5: 121.5 μM or 25.02 mg/L) according to the invention and its metabolites do not have any mutagenic effects on the TA97a, TA98, TA100, TA102 and TA1535 lines.

[0465] Without the activation system S9, the active mixture according to the invention is not mutagenic on the TA102 strain at 0.50 mg/L.

[0466] In this test in vitro, the mutagenic effect of the active mixture and of its metabolites according to the invention is limited for its uses (strain TA102).

[0467] II—Dermato-Sensitization After Induction (Hamster Female; OECD 406, ISO 10993-10: 2013 and ICH Memorandum SCCP 2005)

[0468] According to the protocol of Magnusson & 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 potentialities of the skin of hamster females were studies according to three protocols.

[0469] Protocol 1: Determination of the Tolerated Limiting Dose

[0470] Concentrations of Mixture 1 from 1.25 mg/L to 20.00 mg/L of active substance contained in 3 g/L to 48 g/L of H.sub.2O.sub.2tracer, respectively, in a 1 ml «patch» were applied for 24 h on the flank of the animals, and then the animals were observed.

[0471] The concentration of the Mixture 1 at 10 mg/L of active substance contained in 24 g/L of the tracer is well tolerated and causes moderate irritation.

[0472] The concentration of the Mixture 1 at 5.0 mg/L of active substance contained in 12 g/L of H.sub.2O.sub.2 tracer is well tolerated and is considered as the maximum non-irritating dose.

[0473] Protocol 2: Determination of the Tolerated Limiting Dose After Induction by Subcutaneous Injection.

[0474] Concentrations from 5 mg/L to 10 mg/L of active substance contained in 12 g/L to 24 g/L of the H.sub.2O.sub.2 tracer, respectively, of the Mixture 1 were injected (100 μL) via a subcutaneous route. At 8 days (first challenge), a first topical application as a 1 ml “patch” was achieved. At 27 days (second challenge), a second topical application as a 1 ml «patch» was achieved. The observations were made at 29 days.

[0475] The concentration of the Mixture 1 at 5 mg/L of active substance, contained in 12 g/L of the tracer, is well tolerated and does not produce any irritation.

[0476] Protocol 3: Study of the Irritation Induced by Stimulation and Topical Application of the Mixture 1 at 5 mg/L of Active Substance Contained in 12 g/L of the H.sub.2O.sub.2 Rracer.

[0477] 100 μl of Mixture 1 at 5.0 mg/L was injected via a subcutaneous route. At 4 days, the injected area was stimulated with a solution of sodium dodecyl sulfate (SDS) at 10% before topical application of the Mixture 1 (1 ml) in a “patch”. At 6 days, a second topical application was achieved. At 21 days (challenge) a topical application of the Mixture 1 (1 ml) in a «patch» with concentrations from 6.26 to 10 mg/L of active substance contained in 15 to 24 g/L of tracer was achieved.

[0478] The Mixture 1 at 5.0 mg/L of active substance contained in 12 g/L of tracer is well tolerated and the cutaneous sensitization score by the formulation is equal to 0/3.

[0479] Conclusions: The Mixture 1 at 5.0 mg/L of active substance is not irritating. No macroscopic alteration of the skin of the animals was observed. Accordingly, there is no need of any anatomo-pathological study.

[0480] III—Buccal and vaginal Irritation (Female Rabbit; OECD(99)20, -21, -23 -24, -(95)115, -(02) 9 and ISO-10993-10: 2013)

[0481] The goal of this study was to evaluate the vaginal and buccal tolerance of the Mixture 1 at 5.0 mg/L of active substance contained in 12 g/L of tracer according to an exposure compliant with a 5-day human therapeutic use.

[0482] From day 0 to day 6, every 24h, the animals are treated (1 ml) via a buccal and vaginal route. At day 7, the animals are observed and their weight measured.

[0483] Conclusions: The concentration of the Mixture 1 at 5.0 mg/L of active substance is well tolerated. The buccal and vaginal irritation index is 0/16 and 1/16, respectively. Accordingly, there is no need for an anatomo-pathological study.

[0484] IV—Ocular Irritation Test (ICCVAM ADD B)

[0485] The ocular irritation test used was the substitution test HET-CAM (Hen's Egg Test—Chorioallantoic Membrane), published in the Official Journal dated 26 Dec. 1996. It is recommended by ICCVAM.

[0486] Although this particularly sensitive method is not officially validated by the E.U., it is accepted for a use aiming at locating the even slightly irritating substances including those which would be ocular irritants in order to assign them the label R41 (2002).

[0487] Briefly, the method consists of applying a test substance of the Mixture 1 at 3.75 and 5.00 mg/L of an active substance in 9 and 12 g/L of tracer, respectively, on the chorioallantoic membrane of a fertilized hen egg. At 0.5, 2 and 5 mins post-contact, the three following criteria are observed and scored: hemorrhage, coagulation and hyperemia.

[0488] Results and Conclusions:

[0489] The Mixture 1 at 3.75 mg/L or 5.00 mg/L of active substance may be classified as moderately irritating (Irritation index or IS=6.8±0.4 and 6.3±0/21, respectively), i.e. it induces a slight lysis (severity score=1/3) immediately after its introduction or at 30 s post-contact, no hemorrhage or coagulation.

[0490] Considering the Q-value (ratio of the indices between that of the positive substances of reference NaOH 0.1M [IS=15±3] and 1% SDS [10±2] vs. Mixture 1), Q vs. NaOH=0.51 to 0.54 and Q vs. SDS=1.10 to 1.18 for the Mixture 1 at 3.75 and 5.00 mg/L of active substance, respectively.

[0491] Accordingly, with regard to this test of an extreme sensitivity, which is much more significant than the already highly performing vaginal irritation test in female rabbits, the Mixture 1 is considered as a slightly (3.75 mg/L) to moderately (5.00 mg/L) irritating substance.

[0492] V—Study of the Acute Toxicity by Cutaneous Application and Intravenous Injection—Maximum Tolerated Doses (MTD) and Dose Without any Observable Toxic Effect (NOAEL) (Rat; OECD 474, ICH M3(R2) and S6(R1))

[0493] The goal of this study is to determine the Maximum Tolerated Dose (MTD) of the Mixture 1 by cutaneous application (1 ml) or intra-caudal injection (5 ml/kg). The clinical observations are reported until day 14.

[0494] Conclusions of the Cutaneous Application Protocol:

[0495] The Mixture 1 has a dose of 50 μg/kg of active substance (1 ml to 10 mg/L) which is well tolerated. A slight erythema of the treated area is observed as well as the occurrence of small crusts which disappear within 2 days. No irritation is observed.

[0496] The same proportionally slighter symptoms are observed for an application of the Mixture 1 of 37.5 μg/kg of active substance (1 ml to 7.5 mg/L).

[0497] The Mixture 1 at the doses of 25.0 and 31.3 μg/kg of active substance (1 ml at 5.0 and 6.26 mg/L) is well tolerated. No cutaneous, metabolic or physiological symptom is observed at D14.

[0498] The MTD of the Mixture 1 in a cutaneous application in rat is of 31.3 μg/kg of active substance (1 ml to 6.26 mg/L).

[0499] The NOAEL (dose without any observable toxic effect) of the Mixture 1 in cutaneous application in rats is of 31.3 μg/kg of active substance (1 ml at 6.26 mg/L).

[0500] Conclusions of the Intra-Caudal Injection Protocol:

[0501] The difficulty of intravenous administration of the Mixture 1 does not lie in an intrinsic toxicity of the invention but on its decomposition in contact with blood which notably generates molecular oxygen which may be responsible for embolism. The ethical limiting dose (3 rats) having demonstrated a reversible respiratory arrest is 14.6 μg/kg of active substance (5 ml/kg of the Mixture 1 at 2.92 mg/L). No sign of toxicity was recorded until D14.

[0502] The Mixture 1 is tolerated after intravenous injection at 12.5 μg/kg of active substance (5 ml/kg of the Mixture 1 at 2.5 mg/L). Physiological difficulties (movements and respiratory difficulties) were observed in animals at the moment of the injection. No sign of toxicity was recorded until D14.

[0503] The same symptoms proportionally less significant were observed after an intravenous injection at 10.4 μg/kg of active substance (5 ml/kg of the Mixture 1 at 2.1 mg/L). No clinical sign or modification of the weight curve was reported. No sign of toxicity was recorded until D14. The macroscopic observation of the organs at D14 after autopsy did not reveal anything.

[0504] The MTD of the Mixture 1 in an intra-caudal injection is 12.5 μg/kg of active substance (5 ml/kg of the Mixture 1 at 2.5 mg/L).

[0505] VI—Study of the Genotoxicity In Vivo by Searching for the Generation of Micronuclei after Intravenous Injection of the Mixture 1 (Rat Males and Females; OECD 474 and ICH S2(R1))

[0506] Rat females (n=5) and male rats (n=5) were injected (two treatments at an interval of 22-26 hours; 5 ml/kg) with the HPLC water carrier (intravenous route), the cyclophosphamide positive controls at 5 and 10 mg/kg, intravenous route and ethyl methanesulfonate at 100 and 150 mg/kg (intraperitoneal route) and the Mixture 1 at 1.46, 4.17 and 12.5 μg/kg of active substance (intravenous route).

[0507] The morbidity, the mortality, the weight of the animals and the clinical parameters (temperature, skin, hair, eyes, mucous membranes, secretions and excretions, respiratory and neurological function, gait and posture) were noted. 36 h to 48 h after the second treatment, the blood of the animals was collected and analyzed in flow cytometry (toxicity control: reduction of the proportion of immature erythrocytes (CD-71-positive). The proportion of micro-nucleated immature erythrocytes (reticulocytes) was evaluated on 4,000 observations per blood sample. Each rat population (n=10) for each tested substance represents 40,000 observations (Table 5).

TABLE-US-00018 TABLE 5 Micro- nucleated Reticu- reticu- Substance mg/kg gender n locytes (%) locytes (%) Before # male 5 4.39 ± 0.92 0.15 ± 0.09 treatment female 5 2.60 ± 0.57 0.17 ± 0.07 Negative # male 5 5.17 ± 1.47 0.32 ± 0.31 control female 5 3.82 ± 0.29 0.12 ± 0.04 Water Positive 5 male 5 3.99 ± 0.49 0.56 ± 0.17 control 1 female 5 2.41 ± 0.54 0.40 ± 0.13 Cyclophos- 10 male 5 1.95 ± 0.59 1.62 ± 0.29 phamide female 5 1.30 ± 0.43  1.02 ± 0.26** Positive 100 male 5 1.41 ± 0.32  0.68 ± 0.35** control 2 female 5 1.86 ± 0.25  0.48 ± 0.16** Methane- 150 male 5 1.33 ± 0.34  0.81 ± 0.48** sulfonate female 5 0.45 ± 0.33  0.59 ± 0.26** Mixture 1 1.46 .Math. male 5 5.32 ± 0.76 0.10 ± 0.02 10.sup.−3# female 5 3.35 ± 0.46 0.09 ± 0.03 4.17 .Math. male 5 4.82 ± 0.41 0.11 ± 0.03 10.sup.−3# female 5 3.35 ± 0.83 0.09 ± 0.02 12.5 .Math. male 5-3* 4.79 ± 0.16 0.22 ± 0.12 10.sup.−3# female 5-1* 3.40 ± 0.19 0.09 ± 0.02 *dead animals during the first treatment by embolism due to the release of molecular oxygen from the Mixture 1 in contact with blood. **statistically different from the Control group. .sup.#in active substance MoO.sub.4.sup.2−

[0508] Conclusion: As compared with the control groups (positive and negative), the Mixture 1 does not have any genotoxicity by inducing production of micro-nucleated reticulocytes after intravenous injection in rats (n=10, including 5 males and 5 females) with a range of 1.46, 4.17 and 12.5 μg/kg of active substance.

Example 12—Comparison with Other Compositions

[0509] The present example notably shows the influence of the composition, in particular of the redox potential on:

[0510] 1—the role of calcium in the Fenton/Haber-Weiss “like” catalysis of the tested formulations.

[0511] 2—the existence of a difference between three 5.0 mg/L formulations of active substance.

[0512] S1: Mixture 1,

[0513] S2: composition according to U.S. Pat. No. 6,660,289,

[0514] S3: composition according to WO/2010/004161.

[0515] Protocol 1 (Tables 6 and 7):

[0516] 2 measured parameters: i) pH, ii) redox potential.

[0517] catalyst CaCl.sub.2 (1M, in H.sub.2O): addition in a final concentration of 24 mM

[0518] kinetics: initial (T0), 1, 2, 3 minutes (T1, T2, T3) after adding CaCl.sub.2 with stirring.

[0519] Results and Discussion:

TABLE-US-00019 TABLE 6 Solution S1 T0 CaCl.sub.2 24 mM T1 T2 T3 Average (between T1 and Δ/T0 pH 4.70 ↓ at T0 4.58 4.59 4.59 4.59 over 3 min. −0.11 Redox pot. E° (mV) 359 383 383 383 383 over 3 min. +24 Solution S2 T0 CaCl.sub.2 24 mM T1 T2 T3 Average (between T1 and Δ/T0 pH 2.85 ↓ at T0 2.65 265 2.66 2.65 over 3 min −0.20 Redox pot. E° (mV) 437 479 475 474 476 over 3 min +39 Solution S3 T0 CaCl.sub.2 24 mM T1 T2 T3 Average (between T1 and Δ/T0 pH 3.01 ↓ at T0 2.86 2.87 2.87 2.87 over 3 min. −0.14 Redox pot. E° (mV) 414 465 464 464 464 over 3 min +50

[0520] The pH of the skin is from 4.93+/−0.45 to 5.12+/−0.56. The vaginal pH is normally of the order of 4.50 and may range up to 6.00 at the menopause.

[0521] the solution S1 is buffered and at a pH of the order of the one of the human skin and mucous membranes. This pH remains relatively constant up to 3 mins of contact after adding CaCl.sub.2 final 24 mM. A moderate increase of E° is observed which may partly come from the addition of the chlorine of CaCl.sub.2.

[0522] An acid pH amplifies the cytotoxic lipid peroxidation mediated by iron.

[0523] The solution S2 is not buffered. Its pH is not very compatible for contact with the skin or the mucosas (including the probable destruction of the commensal flora in a therapeutic use). This pH remains relatively constant up to 3 mins of contact after adding CaCl.sub.2 at a final 24 mM. An increase at least less moderate vs. S1 of E° is observed which may partly come from the addition of the chlorine of CaCl.sub.2.

[0524] The solution S3 is not buffered. Its pH is not very compatible for contact with the skin or the mucous membranes (including the probable destruction of the commensal flora in a therapeutic use). This pH remains relatively constant up to 3 mins of contact after adding CaCl.sub.2 final 24 mM. A significant increase (+50 mV) of E° is observed which may partly come from the addition of the chlorine of CaCl.sub.2 but with regard to the difference in potential increase vs. S1 and S2, there would probably not be this single factor.

[0525] As regards the Fe ions in situ, at an acid pH (<2.33), the redox potential is of the order of 400 mV and the Fe in the (III) form, i.e. in its most cytotoxic form. At a pH of the order of 4.5, the redox potential is of the order of 300 mV, the hemoglobin Fe in the (II) form and the plasma Fe complexed with transferrin in the (III) form.

[0526] At the pH of the solutions S2 and S3, the iron oxide form Fe(OH).sub.3 is highly disadvantaged. Accordingly, the Fenton-Haber-Weiss reactions are not very effective.

[0527] The value of the potential of the pair Fe(OH).sub.3/Fe.sup.2+ (Fenton type) is: E′°=1.19−0.18×pH.

TABLE-US-00020 TABLE 7 Act. subst E°.sub.Fe(OH)3/Fe2+ E°.sub.solution (mg/L) pH (mV)* (mV) S1 5.0 4.70 344 354 S2 5.0 2.80 686 428 S3 5.0 3.00 650 418 *in situ cell level with iron

[0528] According to this study, there is confirmation that the pair Fe.sub.(OH)3/Fe.sup.2+ is disadvantaged. Even if the redox potential of this pair is improved (418-428 mV), it remains too significant for cell contact.

[0529] the transmembrane potential of an animal cell is negative (≈−70 mV). The more significant is the oxidation-reduction potential of the solution, the more there will be alteration of the transmembrane potential and more there will be induction of the cell oxidative stress (genic induction of apoptosis, of the eNOS routes, . . . ) with concomitantly induction of a calcium influx which potentializes the significant cell stress already induced by the solutions 2 and 3.

[0530] Accordingly, the pH and the redox potential of formulation S2 and especially the redox potential of the formulation S3 are most likely cytotoxic. Both of these solutions S2 and S3 cannot be suitable for therapeutic use.

[0531] Protocol 2 (Table 8):

[0532] The cell and plasma concentrations of calcium are of the order of 2 to 3mM. This protocol is based on a therapeutic use of the formulations.

[0533] 1 measured parameter: residual amount of peroxides (H.sub.2O.sub.2, peracetic acid; test range Quantofix between 0 and 25 mg/L).

[0534] catalyst CaCl.sub.2 (1M, in H.sub.2O): addition in a final concentration of 1.25, 2.5, 10, 25 and 50 mM.

[0535] final volume: 1 ml=0.950 μL of tested solution+0.05 μL of the CaCl.sub.2 dilution. The dilution factor of the tested solution: 1.05. Final concentration of the tested solution 11.4 g/L (initial 12 g/L) of the tracer H.sub.2O.sub.2 containing 4.76 mg/L of active substance (initial 5.00 mg/L).

[0536] kinetics: 5 minutes after adding CaCl.sub.2 with stirring.

[0537] Results:

TABLE-US-00021 TABLE 8 Test Tested Final [CaCl.sub.2] (mM) [peroxides].sub.final (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 ≈25 6 S2 2.5 >25 7 S3 2.5 >25 8 S3 1.25 ≈25

[0538] The deactivation of the formulation S1 at 5 minutes of contact with CaCl.sub.2 2.5 mM (physiological concentration) corresponds to a peroxide decrease by a factor 1,500 (Test 4), i.e. from 12 g/L to 8 mg/L of tracer and 5.00 mg/L to 3.33 μg/L of active substance).

[0539] Comparatives Conclusions Concerning the Formulations S2 and S3

[0540] The solution S2, regardless of the metal used, is not compatible with a therapeutic application, by:

[0541] its non-bufferable pH for retaining its potentialities,

[0542] its cytotoxic redox potential,

[0543] its relative independence to calcium catalysis,

[0544] its cytotoxicity under physiological conditions due to H.sub.2O.sub.2 and peracetic acid (in addition to the presence of Ag.sup.+ ions, for example, having irreversible cytotoxicity),

[0545] under the experimental conditions used, there is no Fenton-Haber-Weiss like catalysis therefore there is no reactive radical production for therapeutic use.

[0546] The solution S3, regardless of the metal used and the chelating pair described in patent application WO2010/004161, is not compatible with a therapeutic application, by:

[0547] its pH which is not compatible with BAPTA,

[0548] its cytotoxic redox potential,

[0549] its relative independence to calcium catalysis,

[0550] its cytotoxicity under physiological conditions due to H.sub.2O.sub.2 and peracetic acid,

[0551] under the experimental conditions used, there is no Fenton-Haber-Weiss like catalysis, therefore there is no reactive radical production for therapeutic use.

[0552] On the contrary, the reaction chemical model of the formulation S1 according to the invention is correct because of its destabilization by calcium.

[0553] For the formulation S1, the selections:

[0554] of the pH,

[0555] of the buffering pair (addition of a single element of the pair: acetic acid, since peracetic acid is slightly produced in situ),

[0556] of the transition metal and of its valences compatible with the Fenton-Haber-Weiss like reactions or not,

[0557] of the pair of chelating agents (compatibility between them with Kd.sub.Ca2+ and Kd.sub.Mo, Kd.sub.Fe2+/Fe3+ in a “cascade”),

[0558] the non cell penetration of chelating agents,

[0559] of the activation of the complex of the solution S1 by calcium or other existing metal ions in situ, at physiological doses and pH (importance of the selection of the chelating agents), at a redox potential limiting the lipid oxidations notably (little production of Fe(III)) and the instantaneous generation of permeating reactive radicals at a short range, without accumulation, make this formulation compatible with therapeutic use.

[0560] The solution S1 is very strongly degraded at physiological concentrations of calcium with a minimum at 2.5 mM. A reduction (1.25 mM) or an increase (from 10 to 50 mM) of this non-physiological calcium concentration strongly alters this degradation of the peroxides due to the Fenton-Haber-Weiss like reactions by displacement of the equilibria (mass action law). The chemical complex corresponding to the solution S1 is totally adapted to a specific reaction in the cellular and plasma calcium constant.

Example 13—Example of an Active Mixture According to the Invention with a Combination of Mo and La

[0561] This example of composition (“Mixture 2”) according to this invention is made with a molybdenum salt and a lanthanum salt. A composition of the formula according to Table 9 is prepared, expressed as the initial concentration of the components:

TABLE-US-00022 TABLE 9 Substances Mixture of the invention Sodium Molybdate 20.7 μM Lanthanum Nitrate 11.5 μM BAPTA 42 μM EGTA 1051.6 μM H.sub.2O.sub.2 353 mM CH.sub.3COOH/CH.sub.3COONa 70 mM pH (by NaOH) 4.4-5.0 E°.sub.redox 300 to 420 mV

[0562] The composition is called “initial” because it corresponds to the concentrations of the added reagents without taking into account the catalytic process used.

[0563] All the substances used in the synthesis and the finished product are validated by IR-FT spectrum.

TABLE-US-00023 TABLE 10 Molecular Product Purity Formula No CAS Sodium Molybdate dihydrate 98-103%  Na2MoO4•2H2O 10102-40-6 lanthanum Nitrate hexahydrate ≥99% La(NO.sub.3).sub.3•6H.sub.2O 10277-43-7 BAPTA : (1,2-bis(o-aminophenoxy)ethane- ≥98% C22H24N2O10 85233-19-8 N,N,N′,N′-tetraacetic acid) EGTA: (ethylene glycol-bis(2-aminoethylether)- ≥99% C14H24N2O10 67-42-5 N,N,N′,N′-tetraacetic acid) Hydrogen Peroxyde 29-31%  H2O2 722-84-1 Glacial Acetic acid* 99.8-100.5%     CH3COOH 64-19-7 Sodium Acetate trihydrate 99-101%  C2H3NaO2•3H2O 5010524 Sodium hydroxyde 10N NaOH 1310-73-2 Demineralized water   1 μS H2O 7732-18-5

[0564] The active substance is: (MoO.sub.4).sup.1−, Na.sup.+ (lanthanum hydroperoxomolybdate). Molecular Weight: 481.81 g/mol.

[0565] Manufacturing Process:

[0566] The manufacturing process is identical to that of Mixture 1 in Example 1, with the exception of the modifications described below:

[0567] Two SC solutions are being prepared:

TABLE-US-00024 Solution A - Na.sub.2MoO.sub.4•2H.sub.2O 200 mg (final 2.4 mM) (source of Mo(VI); 100%) Water qsp 340 ml Gentle agitation, room temperature Solution B - La(NO.sub.3)3•6H.sub.2O 200 mg (1.4 mM final) (source of La(III); 100%) Water qsp 340 ml Gentle agitation, room temperature

[0568] Solution A (10 mL) and then solution B (10 mL) are gradually introduced into the Si2 solution to prepare an 51 solution of lanthanum hydroperoxomolybdate Step (vii): Adding Chelating Agents—Preparation of an S4 Solution

TABLE-US-00025 Introduction BAPTA (98.8%) 20 mg/L, Introduction EGTA (99.1%) 400 mg/L

[0569] The pH of the final solution is adjusted by NaOH to 4.67

[0570] The final E.sub.redox is: 380 mV

[0571] In Mixture 2, the final concentration of La(MoO.sub.4).sup.1−, Na.sup.+ is: 20.8 μM.

Example 14—Evaluation of the Oxidoreductive Efficiency of the Invention in Double Complex Molybdate and Lanthanate on Human Plasma

[0572] In order to compare the oxidative efficiency of the (MoO.sub.4).sup.2− (Mixture 1) and La(MoO.sub.4).sup.1− (Mixture 2) formulations, formulations with concentrations comparable to those described in FIG. 3 and lower than those described in FIG. 5 were contacted with human plasma for 2 min. After subtraction of the experimental blanks, specifically in this concentration zone, Mixtures 1 and 2 according to the invention generate an increase in the in situ production of oxidized quercetin. The final concentrations of MoO.sub.4.sup.2− and La(MoO.sub.4).sup.1− tested were then: 0.00, 3.80, 7.59, 1215 nM and 0.00, 3.25, 6.50, 1,040 nM, respectively (FIG. 8).

[0573] The difference in oxidized quercetin production (apex around 295 nm) between the two formulations (Mixture 1 and Mixture 2) is not significant. The oxidoreductive efficiency between the two compositions according to the invention is comparable.