New uses of aqueous solutions with high hydrogen concentration

Abstract

The present invention refers to new uses of an aqueous solution comprising molecular hydrogen. In particular, the present invention relates to the use of an aqueous solution comprising molecular hydrogen as an adjuvant therapy in patients undergoing chemotherapy to reduce the side effects associated with the therapy itself.

Description

[0001] “NEW USES OF AQUEOUS SOLUTIONS WITH HIGH HYDROGEN CONCENTRATION” FIELD OF THE INVENTION

[0002] The present invention refers to novel uses of an aqueous solution comprising molecular hydrogen.

[0003] In particular, the present invention relates to an aqueous solution comprising molecular hydrogen, at least one salt, and ascorbic acid or a salt thereof, for use in reducing the side effects associated with anti-cancer therapies, in particular chemotherapeutic drugs. The invention also relates to the use of an aqueous solution comprising molecular hydrogen in the treatment of diseases characterized by inflammatory states.

[0004] STATE OF THE ART

[0005] Tumors are one of the main causes of death in humans and animals.

[0006] The main therapies currently used to treat tumors include surgical removal of the tumor, radiotherapy, and chemotherapy.

[0007] Chemotherapy is a medical therapy aimed at attacking solid and hematological malignant tumors and consists in the administration of different types of drugs that act by destroying tumor cells.

[0008] The choice of drugs to be used and the chemotherapy protocols depend on the general clinical conditions of the patient, his or her age, and the type, stage, and biological characteristics of the tumor.

[0009] The most commonly used chemotherapeutic drugs to date, such as doxorubicin, gemcitabine, and cisplatin, prevent cell multiplication by interfering with the mechanisms related to this process, and in doing so eliminate cancer cells by inducing their death (cytotoxic action).

[0010] Tumor cells are, in fact, characterized by a much faster reproduction cycle than normal cells, so chemotherapy is more effective especially on fast-growing tumors, because it is able to block or slow down their development finally reducing their volume.

[0011] However, all the chemotherapeutic drugs currently used have high cytotoxicity, hepatotoxicity, nephrotoxicity, neurotoxicity and gastrotoxicity.

[0012] This means that chemotherapy also has serious consequences on some types of healthy cells, especially those subject to fast replication, such as the hair follicles, bone marrow, oral mucosa and gastrointestinal mucosa cells.

[0013] This results in very serious side effects associated with chemotherapy treatment that appear, such as for example hair loss, anemia, decreased immune defenses, vomiting, diarrhea, inflammation or infection of the mouth, asthenia, fibromyalgia, excessive weight loss, liver damage and cognitive problems (chemo brain), osteoarticular pain, and neuralgia in the hands and feet. These side effects have an extremely negative impact on the patient’s well-being and quality of life.

[0014] To overcome these serious problems and allow the body to recover from the side effects caused by the therapy itself, physicians limit the intensity, frequency and efficacy of the chemotherapy dose, alternating periods of rest with cycles of therapy. In this way, however, the full therapeutic potential of the chemotherapeutic drugs is inevitably interfered with (Rajagopalan, S. Cancer Res. 1988; Hale, J. P. Arch. Dis. Child 1994; Dobyan, D. C., J. Pharmacol. E.T1980; Fillastre, J. P. Toxicology let 1989).

[0015] When chemotherapy is the only possible therapy to ensure patient’s survival, it is however crucial to fully exploit the therapeutic potential of the drugs used.

[0016] Moreover, limiting the administration of the chemotherapy treatment is not even sufficient to effectively counteract all the side effects associated with the chemotherapeutic drugs.

[0017] To reduce gastrointestinal side effects associated with chemotherapy treatment, the use of thymosin a1 (US6462017B1) is known.

[0018] However, the use of said thymosin a1 is not effective in alleviating other types of side effects associated with chemotherapy treatments.

[0019] Consequently, the need to develop effective methods and drugs to alleviate the side effects of malignant tumors treatment, in particular those resulting from chemotherapy, is still strongly felt.

[0020] Therefore, the aim of the present invention is to provide a therapeutic strategy that is able to reduce side effects associated with the use of drugs and anti-cancer therapies in general, both drugs such as chemotherapy, but also cell therapies, such as for example CAR-T, CAR-NK, radiotherapy or surgical removal.

[0021] SUMMARY OF THE INVENTION

[0022] The inventors have surprisingly found that an aqueous solution comprising molecular hydrogen was able to counteract and therefore reduce side effects associated with antitumor treatments. Advantageously, the aqueous solution comprising molecular hydrogen therefore allowed the maintenance of the therapeutic strategies for treating the tumor itself, since the reduction of side effects allowed the maintenance of the normal therapy, chemotherapy or radiotherapy or cellular, protocols foreseen in the tumor treatment, and improved patient compliance. The present invention therefore concerns an aqueous solution comprising molecular hydrogen, at least one salt, and ascorbic acid or a salt thereof, for use in reducing side effects associated with anti-cancer therapies, in particular with chemotherapy and radiotherapy drugs, wherein said aqueous solution is administered percutaneously or transdermally.

[0023] Advantageously, the solution for the use of the invention has proven to be able to limit the damage caused by the administration of chemotherapeutic agents in oncologic patients, thus allowing the maintenance of antitumor treatment protocols.

[0024] The use of the aqueous solution according to the present invention allows to increase the well-being and improve the quality of life of oncologic patients undergoing anti-cancer therapy, preferably chemotherapy, thanks to a reduction of side effects associated with it. Furthermore, the use of the aqueous solution according to the present invention is safe and effective, and substantially free of side effects.

[0025] Surprisingly and advantageously, the inventors, starting from the treatment of side effects of oncologic patients undergoing chemotherapy, found an anti-inflammatory activity of the solution of the invention when administered percutaneously or transdermally, and therefore realized that the solution object of the present invention was also able to reduce inflammatory processes generated by a diseases selected from the group consisting of pubalgia, arthrosis, tendonitis, tennis elbow, arthritis, muscle tear and strain, fatigue, celiac disease, psoriasis and neuropathy.

[0026] As will be apparent from the experimental part, in fact, the solution of the invention was able to reduce the pro-inflammatory cytokines generated not only by anti-cancer treatments, but present in many diseases with a high inflammatory state.

[0027] Surprisingly, and in a preferred embodiment, the inventors of the present invention have found in oncologic patients an improvement in the state of intestinal dysbiosis, typical of oncologic patients, as a result of the use of the molecular hydrogen solution when administered percutaneously or transdermally, and have understood that they could treat intestinal dysbiosis also in healthy subjects.

[0028] Therefore, in another aspect the invention concerns an aqueous solution comprising molecular hydrogen, at least one salt, and ascorbic acid or a salt thereof, for use in the treatment of intestinal dysbiosis, wherein said aqueous solution is administered percutaneously or transdermally.

[0029] The molecular hydrogen solution of the invention, when administered percutaneously or transdermally, was therefore also able to positively modulate altered microbiota in a healthy subject.

[0030] Therefore, the invention also consists in the cosmetic use of an aqueous solution comprising molecular hydrogen, at least one salt, and ascorbic acid or a salt thereof, in the positive modulation of the intestinal microbiota, wherein said aqueous solution is administered percutaneously or transdermally.

[0031] In fact, the inventors observed that the administration of the solution of the invention, when administered percutaneously or transdermally, allowed to promote the growth of microorganisms with a positive impact on the microflora, thus restoring a correct balance of the intestinal microbiota.

[0032] Advantageously, the use of the solution of the invention proved to be a concomitant treatment to known anti-inflammatory treatments against diseases characterized by high levels of inflammation.

[0033] In another aspect, therefore, the invention relates to a combined use of the solution of the invention and at least one anti-inflammatory drug in the treatment of diseases characterized by high levels of inflammation.

[0034] The administration of the solution for the uses according to the present invention occurs percutaneously or transdermally.

[0035] In another aspect, the invention concerns an aqueous solution comprising molecular hydrogen, sodium hydrogencarbonate, potassium hydrogencarbonate, magnesium chloride and ascorbic acid or a salt thereof, wherein the weight ratio of sodium hydrogencarbonate, potassium hydrogencarbonate, magnesium chloride, and ascorbic acid or a salt thereof is in the range of 0:0:1 :1 to 3:3:3:3 provided that

[0036] - at least one of sodium hydrogencarbonate and potassium hydrogencarbonate is present and wherein when ascorbic acid is present, the solution comprises

[0037] - sodium bicarbonate, potassium hydrogencarbonate, magnesium chloride and ascorbic acid in a weight ratio of 0:1 :3:3 (solution L); or

[0038] - sodium hydrogencarbonate, potassium hydrogencarbonate, magnesium chloride and ascorbic acid in a weight ratio of 2:2:3:2 (solution M), or

[0039] - sodium hydrogencarbonate, potassium hydrogencarbonate, magnesium chloride and ascorbic acid in a weight ratio of 0:3:3:1 (solution N). DESCRIPTION OF THE FIGURES

[0040] Figure 1 shows the results of Example 3, i.e. the percentage of reduction of cytokines IL-6 and TNF-alpha in oncologic patients.

[0041] Figure 2 shows the results of Example 4 relating to the reduction of chemotherapy-induced hepatotoxicity by the invention.

[0042] Figure 3 shows the results of Example 5 relating to the positive modulation action of intestinal microbiota of healthy subjects by the solution of the invention.

[0043] DETAILED DESCRIPTION OF THE INVENTION

[0044] The present invention therefore concerns an aqueous solution comprising molecular hydrogen, at least one salt, and ascorbic acid or a salt thereof, for use in reducing the side effects associated with anti-cancer therapies, in particular chemotherapy and radiotherapy drugs, wherein said aqueous solution is administered percutaneously or transdermally.

[0045] Advantageously, the solution for use of the invention has proven to be able to limit the damage caused by the administration of chemotherapeutic agents in oncologic patients, thus allowing the maintenance of antitumor treatment protocols.

[0046] The use of the aqueous solution according to the present invention allows to increase the well-being and improve the quality of life of oncologic patients undergoing anti-cancer therapy, preferably chemotherapy, thanks to a reduction of the side effects associated therewith. Furthermore, the use of the aqueous solution according to the present invention is safe and effective, and substantially free of side effects.

[0047] When the term ‘‘molecular hydrogen” is used in the present invention, it is intended to mean hydrogen gas dissolved in water and present in the form of bubbles in micro- and nanoamounts.

[0048] Molecular hydrogen is preferably present in the solution in an amount (concentration) in the range from 0.3 ppm (mg / L) to 3.6 ppm (mg / L), more preferably from 0.4 ppm (mg / L) to 1.2 ppm (mg / L), even more preferably from 0.5 ppm (mg / L) to 0.7 ppm (mg / L), advantageously of about 0.6 ppm (mg / L).

[0049] Preferably, according to the invention, the at least one salt is selected from the group consisting of NaCI (sodium chloride); NaHCOs (sodium bicarbonate); Na2[CO3] (sodium carbonate); NaaCeHsO? (sodium citrate); NaHPCh (sodium dihydrogen phosphate); CH2(COONa)2 (disodium malonate); KOI (potassium chloride); KHCO3 (potassium bicarbonate); K2[CO3] (potassium carbonate); K3C6H5O7 (potassium citrate KHPO3; K2PO3 (potassium hydrogen phosphate); KOCOCH(OH)CH(OH)COOH (potassium tartrate monobasic); KOCOCH(OH)CH(OH)COK (potassium tartrate);

[0050] KOCOCH(OH)CH(OH)COONa (sodium potassium tartrate); MgCfe (magnesium chloride); MgO (magnesium oxide); Mg(OH)2 (magnesium hydroxide); Mg[HCO3]2 (magnesium bicarbonate); MgCOs (magnesium carbonate); MgsfCeHsOzk (magnesium citrate); MgCioH^NaOe (magnesium pidolate); MgSO4 (magnesium sulfate); Mg(HSC>4)2 (magnesium hydrogen sulfate); CaCfe (calcium chloride); Ca[HCO3]2 (calcium bicarbonate); CaCOs (calcium carbonate); CasfCeHsOzh (calcium citrate); CaHPO4 (calcium hydrogen phosphate); Ca(H2PO4)2 (calcium dihydrogen phosphate) and mixtures thereof.

[0051] Said at least one salt is preferably hydrogencarbonate (bicarbonate) of an alkali or alkaline earth metal or chloride of an alkali or alkaline earth metal.

[0052] More preferably, said at least one salt is hydrogencarbonate (bicarbonate) of an alkali metal or chloride of an alkali metal.

[0053] Even more preferably, said at least one salt is selected from the group consisting of sodium hydrogencarbonate (bicarbonate), potassium hydrogencarbonate (bicarbonate) and magnesium chloride.

[0054] Said at least one salt of the invention is preferably present in the aqueous solution in an amount in the range from 10 mM to 500 mM, more preferably from 50 mM to 350 mM.

[0055] According to a particularly preferred embodiment of the present invention, said aqueous solution comprises at least two salts.

[0056] Preferably, according to the invention, said at least two salts are selected from the group consisting of NaCI (sodium chloride); NaHCOs (sodium bicarbonate); Na2[COs] (sodium carbonate); NasCeHsOz (sodium citrate); NaHPOs (sodium dihydrogen phosphate); CH2(COONa)2 (disodium malonate); KCI (potassium chloride); KHCO3 (potassium bicarbonate); fofCOs] (potassium carbonate); K3C6H5O7 (potassium citrate KHPO3; K2PO3 (potassium hydrogen phosphate); KOCOCH(OH)CH(OH)COOH (potassium tartrate monobasic); KOCOCH(OH)CH(OH)COK (potassium tartrate);

[0057] KOCOCH(OH)CH(OH)COONa (sodium potassium tartrate); MgCh (magnesium chloride); MgO (magnesium oxide); Mg(OH)2 (magnesium hydroxide); Mg[HCOs]2 (magnesium bicarbonate); MgCOs (magnesium carbonate); MgsfCeHsOzk (magnesium citrate); MgCioHi2N20e (magnesium pidolate); MgSO4 (magnesium sulfate); Mg(HSO4)2 (magnesium hydrogen sulfate); CaCfe (calcium chloride); Ca[HCO3]2 (calcium bicarbonate); CaCOs (calcium carbonate); CasfCeHsOzh (calcium citrate); CaHPO4 (calcium hydrogen phosphate); Ca(H2PO4)2 (calcium dihydrogen phosphate) and mixtures thereof. Said at least two salts are preferably hydrogencarbonate (bicarbonate) of an alkali or alkaline earth metal and chloride of an alkali or alkaline earth metal, preferably of an alkali metal, even more preferably they are hydrogencarbonate (bicarbonate) of an alkali metal and magnesium chloride.

[0058] In a preferred and advantageous embodiment of the present invention, said at least two salts are preferably sodium or potassium bicarbonate and magnesium chloride.

[0059] Ascorbic acid can be present in the form of a salt or as an acid. When in the form of a salt, it is preferably an ascorbate of an alkali or alkaline earth metal, more preferably of an alkali metal. In an advantageous and even more preferred embodiment of the invention, said salt is sodium ascorbate or potassium ascorbate or mixtures thereof.

[0060] Ascorbic acid, or a salt thereof, is preferably present in an amount in the range from 10 mM to 500 mM, more preferably from 50 mM to 350 mM.

[0061] The aqueous solution of the invention has an ionic strength expressed as a measure of TDS (Total Dissolved Solids, ppm) of between 300 ppm and 9500 ppm, preferably 500 ppm and 9000 ppm, more preferably 8500 ppm.

[0062] The aqueous solution of the invention preferably has an ionic strength in the range from 0.098 mol / L to 0.25 mol / L, preferably from 0.2 mol / L to 0.24 mol / L, more preferably of about 0.220 mol / L.

[0063] Preferred and advantageous embodiments of the ratio between the salts and ascorbic acid, or a salt thereof, of the solution for use according to the invention are:

[0064] - sodium bicarbonate, potassium bicarbonate, magnesium chloride and ascorbic acid, in a weight ratio of 1:1:1 :1 (solution A);

[0065] - sodium bicarbonate, potassium bicarbonate, magnesium chloride and ascorbic acid, in a weight ratio of 2:2:2:2 (solution B);

[0066] - sodium bicarbonate, potassium bicarbonate, magnesium chloride and ascorbic acid, in a weight ratio of 3: 1:3:2 (solution E);

[0067] - sodium bicarbonate, potassium bicarbonate, magnesium chloride and ascorbic acid, in a weight ratio of 1:2:3: 1 (solution F);

[0068] - sodium bicarbonate, potassium bicarbonate, magnesium chloride and ascorbic acid, in a weight ratio of 1 :2:3:3 (solution G);

[0069] - sodium bicarbonate, potassium bicarbonate, magnesium chloride and ascorbic acid, in a weight ratio of 2: 1:3:2 (solution H); - sodium bicarbonate, potassium bicarbonate, magnesium chloride and ascorbic acid, in a weight ratio of 0:1 :2:1 (solution I);

[0070] - sodium bicarbonate, potassium bicarbonate, magnesium chloride and ascorbic acid, in a weight ratio of 0:3:4:2 (solution J);

[0071] - sodium bicarbonate, potassium bicarbonate, magnesium chloride and ascorbic acid, in a weight ratio of 3:0:3: 1 (solution K);

[0072] - sodium bicarbonate, potassium bicarbonate, magnesium chloride and ascorbic acid, in a weight ratio of 0: 1:3:3 (solution L);

[0073] - sodium bicarbonate, potassium bicarbonate, magnesium chloride and ascorbic acid, in a weight ratio of 2:2:3:2 (solution M), and

[0074] - sodium bicarbonate, potassium bicarbonate, magnesium chloride and ascorbic acid, in a weight ratio of 0:3:3: 1 (solution N).

[0075] The aqueous solution of the invention may also comprise other ingredients / substances, such as water-soluble vitamins such as vitamins of the B group, including folic acid, vitamin H, vitamin PP.

[0076] The aqueous solution of the invention preferably has a pH value in the range from 6 to 11 . The administration of the solution for the uses according to the present invention occurs percutaneously or transdermally.

[0077] The percutaneous or transdermal administration route of the use according to the invention allows an effective permeation of hydrogen in the treated organism in a safe manner and without side effects. The presence of salts according to the invention in the solution contributes decisively to a greater permeation of hydrogen, as will be apparent from the experimental part.

[0078] Side effects of anti-cancer therapies preferably comprise, but are not limited to, at least one of reduction of nausea, headache, hair loss, anemia, decreased immune defenses, vomiting, diarrhea, inflammation or infection of the mouth, insomnia, asthenia, fibromyalgia, excessive weight loss, liver damage, cognitive problems (chemo brain) and musculoskeletal pain.

[0079] In one embodiment of the present invention, the administration of the solution according to the present invention occurs before the start of chemotherapy treatments. The administration may occur in one treatment with a determined dose or more treatments with a determined dose in the days preceding the start of chemotherapy therapy. In a preferred embodiment, the administration occurs for the period of anti-cancer therapy with one or more treatments with a determined dose, depending on the severity of the side effects themselves.

[0080] Without being tied to any theory, the inventors observed that percutaneous or transdermal administration before the start of anti-cancer treatments allows for greater efficacy of the anti-cancer therapy itself, and a reduction of its side effects, both in their severity and in their duration.

[0081] In another embodiment of the present invention, the administration of the solution according to the present invention occurs during and / or after the therapy cycle, in single treatments or multiple treatments.

[0082] In a preferred embodiment, the administration occurs with multiple treatments.

[0083] According to the invention, administration to the oncologic patient may be carried out by immersing the body or parts thereof in tanks or devices capable of containing the solution of salts, ascorbic acid or a salt thereof, and molecular hydrogen according to the invention. Among the parts of the body, it is preferable to immerse hands and feet in the solution of the invention. The administration route of the invention has proven to be particularly advantageous for effective permeation of hydrogen into the body.

[0084] Furthermore, hydrogen absorption is promoted and positively modulated by the salts present in the solution. Only the presence in the aqueous solution of a combination of salts according to the invention and hydrogen allows an effective permeation and absorption of hydrogen, as will be apparent from the experimental part.

[0085] Absorption occurs through immersion of hands or feet, or hands and feet, in the solution according to the present invention for a time in the range from 15 to 60 minutes, in a single cycle or several short cycles of 10-15 minutes each.

[0086] The aqueous solution of the invention is preferably at a temperature comprised between 30°C and 47°C, when used in a medical treatment of the invention.

[0087] The use of the solution of the present invention, allowing to reduce the side effects of anticancer therapies, increases the well-being and improves the quality of life of oncologic patients.

[0088] Surprisingly and advantageously, the inventors, starting from the treatment of side effects of oncologic patients undergoing chemotherapy, found an anti-inflammatory activity of the treatment with the solution of the invention, and therefore realized that the use of the solution object of the present invention was able to reduce inflammatory processes generated by and / or consequent to a disease selected from the group consisting of arthritis, arthrosis, rheumatoid arthritis, fatigue, neuropathy, even induced by drugs, celiac disease, psoriasis, tendonitis, tennis elbow, pubalgia, muscle strain and sprain.

[0089] Without being tied to any theory, therefore, the inventors believe that the activity against the side effects of anti-cancer therapies by the solution of the invention is an anti-inflammatory activity that allows to treat also the inflammatory states of a disease selected from the group consisting of arthritis, arthrosis, rheumatoid arthritis, fatigue, neuropathy, even induced by drugs, celiac disease, psoriasis, tendonitis, tennis elbow, pubalgia, muscle strain and strain. All the preferred conditions of the use in the treatment of the side effects generated by anticancer therapies are applicable to the anti-inflammatory use against the diseases reported herein and will not be repeated here.

[0090] Surprisingly, and in a preferred embodiment, the inventors of the present invention found in oncologic patients an improvement in the state of intestinal dysbiosis, typical of oncologic patients, following the use of the molecular hydrogen solution, and have realized that they could treat dysbiosis also in healthy subjects.

[0091] Therefore, in another aspect, the invention concerns an aqueous solution comprising molecular hydrogen, at least one salt, and ascorbic acid or a salt thereof, for use in the treatment of intestinal dysbiosis, when the solution is administered percutaneously or transdermally.

[0092] All the preferred use conditions in the treatment of side effects generated by anti-cancer therapies are applicable to the use in the treatment of intestinal dysbiosis and will not be repeated here.

[0093] The molecular hydrogen solution of the invention was therefore also able to positively modulate the altered intestinal microbiota in a healthy subject.

[0094] Therefore, the invention also consists in the cosmetic use of an aqueous solution comprising molecular hydrogen, at least one salt, and ascorbic acid or a salt thereof, in the positive modulation of the intestinal microbiota, when the solution is administered percutaneously or transdermally.

[0095] In fact, the inventors observed that the administration of the solution of the invention allowed to promote the growth of positive microorganisms thus creating a correct balance of the intestinal microbiota, through the administration of the aqueous solution percutaneously or transdermally.

[0096] Without being tied to any theory, and as demonstrated by the experimental part, the inventors believe that the specific compounds of the aqueous solution of the invention allow to provide a high concentration of molecular hydrogen that permeates the skin by percutaneous or transdermal administration, and is able to intervene on the intestinal microbiota by positively modulating its bacterial composition and also treating intestinal dysbiosis.

[0097] Numerous research and studies have in fact demonstrated that a healthy microbiota guarantees beneficial effects to the entire organism. In particular, a healthy microbiota provides protection against pathogens growth, regulates metabolism and sense of satiety, protects the cardiovascular system, strengthens the host’s immune system, eliminates toxins, promotes the correct digestive process, and actively contributes to the biosynthesis of various vitamins, in particular vitamin B12. As will be apparent in the experimental part, the solution of the invention was able to effectively treat the intestinal microbiota in a positive way.

[0098] In another aspect, the invention concerns an aqueous solution comprising molecular hydrogen, sodium hydrogencarbonate, potassium hydrogencarbonate, magnesium chloride and ascorbic acid or a salt thereof, wherein the weight ratio of sodium hydrogencarbonate, potassium hydrogencarbonate, magnesium chloride and ascorbic acid, or a salt thereof, is in the range from 0:0:1 :1 to 3:3:3:3 provided that

[0099] - at least one of sodium hydrogencarbonate and potassium hydrogencarbonate is present and wherein when ascorbic acid is present, the solution comprises

[0100] - sodium bicarbonate, potassium hydrogencarbonate, magnesium chloride and ascorbic acid in a weight ratio of 0:1 :3:3 (solution L); or

[0101] - sodium hydrogencarbonate, potassium hydrogencarbonate, magnesium chloride and ascorbic acid in a weight ratio of 2:2:3:2 (solution M), or

[0102] - sodium hydrogencarbonate, potassium hydrogencarbonate, magnesium chloride and ascorbic acid in a weight ratio of 0:3:3:1 (solution N).

[0103] Various embodiments and aspects of the present invention, as outlined above and as claimed in the claims section below, are experimentally supported by the following examples.

[0104] EXAMPLES

[0105] Example 1. F of the solution of the invention Different aqueous solutions were prepared using the following ingredients:

[0106] Aqueous solution A: 15g of sodium bicarbonate; 15g of potassium bicarbonate, 15g of magnesium chloride and 15g of ascorbic acid (weight ratio 1 :1 :1 :1 )

[0107] Aqueous solution B: 30g of sodium bicarbonate; 30g of potassium bicarbonate, 30g of magnesium chloride and 30g of ascorbic acid (weight ratio 2:2:2:2)

[0108] Aqueous solution E: 45g of sodium bicarbonate; 15g of potassium bicarbonate, 45g of magnesium chloride and 30g of ascorbic acid (weight ratio 3:1 :3:2)

[0109] Aqueous solution F: 15g of sodium bicarbonate; 30g of potassium bicarbonate, 45g of magnesium chloride and 15g of ascorbic acid (weight ratio 1 :2:3:1 )

[0110] Aqueous solution G: 15g of sodium bicarbonate; 30g of potassium bicarbonate, 45g of magnesium chloride and 45g of ascorbic acid (weight ratio 1 :2:3:3)

[0111] Aqueous solution H: 30g of sodium bicarbonate; 15g of potassium bicarbonate, 45g of magnesium chloride and 30g of ascorbic acid (weight ratio 2:1 :3:2)

[0112] Aqueous solution I: 15g of sodium bicarbonate; 30g of magnesium chloride and 15g of ascorbic acid (weight ratio 1 :0:2:1 )

[0113] Aqueous solution J: 45g of potassium bicarbonate, 60g of magnesium chloride and 30g of ascorbic acid (weight ratio 0:3:4:2)

[0114] Aqueous solution K: 45g of sodium bicarbonate; 45g of magnesium chloride and 15g of ascorbic acid (weight ratio 3:0:3: 1)

[0115] Aqueous solution L: 15g of potassium bicarbonate, 45g of magnesium chloride and 45g of ascorbic acid (weight ratio 0:1 :3:3)

[0116] Aqueous solution M: 30g of sodium bicarbonate; 30g of potassium bicarbonate, 45g of magnesium chloride and 30g of ascorbic acid (weight ratio 2:2:3:2)

[0117] Aqueous solution N: 45g of potassium bicarbonate, 45g of magnesium chloride and 15g of ascorbic acid (weight ratio 0:3:3: 1).

[0118] The salts were added into a container containing water in an amount of 5 L, thus obtaining the solution above.

[0119] Gaseous hydrogen was then introduced into the container, thus obtaining an amount of molecular hydrogen comprised between 0.4 ppm and 1.2 ppm.

[0120] The final pH of the solutions was in the range from 7 to 9, as measured with a VioLab benchtop pH meter, specifically:

[0121] Aqueous solution A: the pH was 7.7 before the introduction of H2 and 8.6 after the introduction of H2 Aqueous solution B: the pH was 7.88 before the introduction of H2 and 8.2 after the introduction of H2

[0122] Aqueous solution E: the pH was 7.9 before the introduction of H2 and 7.9 after the introduction of H2

[0123] Aqueous solution F: the pH was 7.9 before the introduction of H2 and 8.4 after the introduction of H2

[0124] Aqueous solution G: the pH was 7.5 before the introduction of H2 and 8.4 after the introduction of H2

[0125] Aqueous solution H: the pH was 7.8 before the introduction of H2 and 8.4 after the introduction of H2

[0126] Aqueous solution I: the pH was 7.7 before the introduction of H2 and 7.7 after the introduction of H2

[0127] Aqueous solution J: the pH was 7.9 before the introduction of H2 and 8.4 after the introduction of H2

[0128] Aqueous solution K: the pH was 7.3 before the introduction of H2 and 8.6 after the introduction of H2

[0129] Aqueous solution L: the pH was 7.1 before the introduction of H2 and 8.5 after the introduction of H2

[0130] Aqueous solution M: the pH was 8.1 before the introduction of H2 and 9 after the introduction of H2

[0131] Aqueous solution N: the pH was 8.1 before the introduction of H2 and 9 after the introduction of H2

[0132] The ionic strength (I) of the individual solutions was measured immediately upon addition of the salts and after the introduction of H2 by direct measurement of the total dissolved solids (TDS) solutions using the S700 conductivity meter (Mettler Toledo), and the results were expressed in parts-per-million (ppm)

[0133] The relationship between TDS and Ionic Strength follows the conversion equation

[0134] I (mol / L) « 2.5 10'5xTDS (mg / L).

[0135] Data are reported in Tablel : Table 1

[0136] The ionic strength of the solution, as expressed in mol / L after 20 minutes of hydrogen, is

[0137] Solution A: 0.098 mol / L

[0138] Solution B: 0.235 mol / L Solution E: 0.248 mol / L

[0139] Solution F: 0.220 mol / L

[0140] Solution G: 0.225 mol / L

[0141] Solution H: 0.249 mol / L

[0142] Solution I: 0.100 mol / L Solution J: 0.250 mol / L

[0143] Solution K: 0.220 mol / L

[0144] Solution L: 0.218 mol / L

[0145] Solution M: 0.235 mol I L

[0146] Solution N: 0.250 mol / L Example 2

[0147] The solutions prepared in Example 1 were used and tested to evaluate the molecular hydrogen passed through by percutaneous administration of the subjects.

[0148] Two subjects (a man and a woman) were asked to immerse their feet and hands in different containers containing the different prepared solutions (Table 2), and the hydrogen permeated was measured in the exhaled breath of the two subjects by means of an oral respiratory breath-test. The measurements were made before and after immersing the hands and feet, to measure the AH2. The permeation of hydrogen, for the same solutions (Table 2) is a subjective phenomenon linked to the chemical-biological characteristics that make up the exposed tissue.

[0149] The results are reported in the following Table 2. Table 2

[0150] As apparent from the above, the solutions of the invention ensured the passage of molecular hydrogen to the body through percutaneous administration. The best results were obtained with solutions B, E, F, J and H, with a very high amount of molecular hydrogen. As explained above, the inventors believe that the presence of salts and ascorbic acid, or a salt thereof, in an aqueous solution ensures the stability of the hydrogen aqueous solution and allows the transport of molecular hydrogen to the cells through percutaneous administration. It was apparent that in the solutions with a high concentration of molecular hydrogen, but in the absence of the essential ingredients of the solution of the invention, the molecular hydrogen transported to the cells was evidently lower than or equal to the control.

[0151] Below, the results of the amount of H2 measured by the breath test on subjects 1 and 2 after percutaneous administration of the solution of the invention are reported. Specifically, the evaluation was carried out with

[0152] - water only;

[0153] - water + H2;

[0154] - water + solution N;

[0155] - water + solution K;

[0156] - water + H2 + solution N, and

[0157] - water + H2 + solution K.

[0158] The aqueous solution K was: 45 g sodium bicarbonate; 45 g magnesium chloride and 15 g ascorbic acid (weight ratio3:0:3:1); The pH was 7.3 before the introduction of H2 and 8.2 after the introduction of H2.

[0159] The aqueous solution N was: 45 g potassium bicarbonate, 45 g magnesium chloride and 15 g ascorbic acid (weight ratio of 0:3:3:1 ); The pH was 8.1 before the introduction of H2 and 9 after the introduction of H2.

[0160] Table 3 It is apparent from Table 3 that the solutions K and N of the invention, through percutaneous administration, surprisingly allowed the passage of H2 through the cells.

[0161] Example 3: Use of the solution of the invention in the treatment of qastrointestinal side effects in

[0162] Method 0

[0163] To demonstrate the efficacy of the aqueous solution of the present invention in reducing gastrointestinal side effects in oncologic patients, the plasma trend of pro-inflammatory cytokines was evaluated.

[0164] A group of 15 oncologic patients with metastatic tumor at the gastrointestinal level (intestine, liver, kidneys, pancreas, biliary tract, stomach) were treated with the solution of the present invention.

[0165] The oncologic patients were undergoing chemotherapy treatment with Cis-Platinum in combination with Gemcitabine.

[0166] The treatment with the solution of the invention K for a time of 20 minutes per day for 4 weeks. The administration of the solution of the invention also occurred between one chemotherapy infusion cycle and the next for 3 days per week.

[0167] The evaluation involved blood sampling at the beginning and after chemotherapy.

[0168] The levels of tumor necrosis factor a (TNF-a) and IL-6 in the blood of the patients were determined using a Human Proinflammatory Cytokine Multiplex ELISA Kit (Arigo Biolaboratories) according to the manufacturer’s instructions. Plasma was collected using EDTA as an anticoagulant. Plasma samples were collected from the patients.

[0169] The samples were then centrifuged for 10 minutes at 1400 ref and supernatants were aliquoted and stored at -20°C.

[0170] 100 pL of standard mix of the kit and samples were added into wells and any target cytokines present were bound by the immobilized specific antibodies. After 2 hours of incubation at room temperature, the mixed antibody conjugate was added to each well and incubated for 1 hour. After washing off any unbound substances, the HRP conjugate was added to the wells and incubated. The wells were washed thoroughly to remove all unbound HRP conjugate. A TMB substrate was added to the wells and a color developed proportionally to the amount of target cytokines in the initial step. Color development was stopped by adding STOP solution and the color intensity was measured at an O.D. wavelength of 450 nm by SPARK, a multifunctional microplate reader platform. The concentration of human pro-inflammatory cytokines in the samples was then determined by comparing the 450 nm O.D. of samples to the standard curve.

[0171] Results

[0172] Plasma cytokine analysis showed a reduction in the cytokine IL-6 (plasma reference limit 0- 6 pg / mL) in all patients in the range of 10-90% depending on the degree of metastasis and type of chemotherapy, and a consistent reduction in the cytokine TNFa (plasma reference limit 0-8 pg / mL) in the range of 35-50% in all patients (Figure 1).

[0173] Therefore, the treatment with the solution of the present invention showed a direct reduction in the production of tumor pro-inflammatory cytokines IL-6 and TNFa in all metastatic patients with also the concomitant benefit of improving health conditions.

[0174] In fact, all treated patients showed a decrease in side effects associated with chemotherapeutic drugs, such as reduction in nausea, vomiting, headache, fatigue, sense of exhaustion, insomnia, musculoskeletal pain.

[0175] Some patients who were approaching anti-cancer therapy for the first time did not lose their hair during chemotherapy, while those who had lost their hair with the first chemotherapy cycles showed regrowth when treated with the solution of the invention, even concomitantly with the anti-cancer cycle, with concomitant activation and lengthening of the Anagen phase.

[0176] Advantageously, the treatment with the solution of the invention allowed to respect the time schedules of the chemotherapy infusion protocols, thus accelerating healing.

[0177] Example 4: Reduction of chemotherapy-induced hepatotoxicity

[0178] Many drugs frequently cause an increase in liver enzymes (alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase). In particular, chemotherapeutic drugs, such as methotrexate, 5-fluorouracil, irinotecan, tamoxifen and l-asparaginase, Cis Platinum and derivatives, may develop a hepatotoxic phenotype known as “CASH” standing for “Chemotherapy-induced Acute Steatohepatitis”. The mechanism of toxicity is essentially based on mitochondrial toxicity (Meunier L, Larrey D. Chemotherapy-associated steatohepatitis. Ann Hepatol. 2020 Nov-Dec; 19(6):597-601 ).

[0179] Liver damage often forces the patient to interrupt therapy with serious consequences on the regression of the tumor disease. Having a complementary treatment available that can reduce and / or contain the liver damage from chemotherapy is mandatory.

[0180] Analysis method A group of 15 oncologic patients with metastatic tumor at the gastrointestinal level (intestine, liver, kidneys, pancreas, biliary tract, stomach) was subjected to treatment with the solution of the present invention.

[0181] The oncologic patients were undergoing chemotherapy with Cis-Platinum in combination with Gemcitabine for 4 weeks.

[0182] The treatment with solution K of the invention occurred by immersing hands and feet for 20 minutes a day for 2 weeks. The administration of the solution of the invention also occurred between one chemotherapy infusion cycle and the next for 3 days a week.

[0183] The evaluation involved blood sampling at the beginning and after chemotherapy.

[0184] The patients were divided into 3 groups, A, B and C. Group A is the one that showed high liver toxicity at recruitment. Group B showed moderate liver toxicity at recruitment, while group C showed no apparent liver toxicity at recruitment. The parameters considered normal are included in the range 0-35 ll / L.

[0185] The patients were subjected to blood sampling at the beginning and after chemotherapy and the plasma analyzed for ALT (alanine aminotransferase) values and expressed in (U / L) according to the surveillance routine of the oncology clinic.

[0186] Results

[0187] The variations of ALT showed an efficacy of the treatment with the solution of the present invention in all groups (A-C) subjected to chemotherapy.

[0188] With reference to Figure 2, showing the results on patients, the following was observed.

[0189] In group A (high toxicity at recruitment) the treatment with the solution of the present invention drastically reduced liver toxicity in a constant manner even in the presence of chemotherapy infusion, until total normalization. For group B, the typical trend of toxicity induced by treatment after infusion is observed. The treatment contained liver damage, subsequently normalizing ALT even in the presence of drug infusion until normalization. Group C, although showing few ALT peaks, shows a fluctuating trend but still controllable with the use of percutaneous hydrogen.

[0190] In conclusion, the percutaneous treatment with the solution of the present invention has proven to be able to reduce and contain liver damage in the presence of therapy with oncological drugs.

[0191] Example 5: Positive modulation of the intestinal microbiota of healthy subjects

[0192] Clinical protocol and Method To demonstrate the efficacy of the aqueous solution of the present invention in modulating the intestinal microbiota, a group of 14 healthy subjects were subjected for one week to treatment with solution A, L and K of Example 1, which were administered percutaneously by immersing hands and feet in the solution of the invention for 20 minutes a day for 15 days, starting with Solution A for the first 2 days, Solution L for the following 2 days, and finally Solution K for one day. Stool (5 g) was collected before the start of the sessions.

[0193] The collection of fecal samples was carried out using a special sterile container. The sample, if not directly analyzed, was stored at -80°C.

[0194] Phylogenetic and taxonomic analyses were as follows.

[0195] Phylogenetic characterization of the microbiota was performed by next-generation sequencing of the 16S rRNA gene. The analysis was performed by amplification of the hypervariable regions V3 and V4 of the bacterial gene encoding the 16S rRNA, following the Illumina protocol for library preparation. Sequencing was performed on the Illumina MiSeq platform. The sequences obtained were analyzed using the open source software Quantitave Insights Into Microbial Ecology 2 (QIIME 2, Bolyen et al., Nat Biotechnol 2019). The sequences were filtered by quality, grouped into Amplicon Sequence Variants and then assigned to a bacterial taxon.

[0196] High-resolution taxonomic and functional characterization of the microbiota was performed by metagenome shotgun sequencing. Metagenome shotgun sequencing was performed on a subset of samples (selected based on 16S rRNA gene sequencing data) in order to obtain functional and taxonomic information at the species level, including eukaryotes (fungi and parasites) and viruses (including bacteriophages). Indexed libraries were then built using commercial kits and following established pipelines, and sequenced on the Illumina NextSeq platform to obtain > 2 Gbp of high-quality sequences (Q > 30) per sample. Specieslevel profiling was achieved by characterizing metagenomic species with assembly procedures, functional assignment methods, and taxa binning tools from the Meta-Wrap pipeline. Virome and mycobiome were characterized using dedicated pipelines, such as ViromeScan and HumanMycobiomeScan. Strain profiling was performed using StrainPhlAn. The data thus obtained were analyzed using R software for statistical evaluations.

[0197] Results Stool analysis after treatment with the solution of the present invention showed a change in the compositions of the intestinal microbiota, detected by quantification of Procteobacteria, Firmicutes and Faecalibacterium.

[0198] Proteobacteria include a large group of parasitic pathogens, such as Escherichia coli, Salmonella typhi, Vibrio cholerae, Helicobacter pylori and many others. An increased prevalence of Proteobacteria is a potential diagnostic signature of dysbiosis and disease risk; therefore, therapies and supplements and / or tools capable of reducing their amount represent a step forward in activities supporting health and longevity.

[0199] In patients treated with the solution of the present invention, a decrease in the amount of Proteobacteria was detected.

[0200] Firmicutes are a large population of bacteria present in our intestine that show similar characteristics and properties. These microorganisms are characterized by their ability to obtain nourishment from fats and sugars that are normally taken with the diet, helping the body to better assimilate them. In patients treated with the solution of the present invention, an increase in Firmicutes was detected.

[0201] Faecalibacterium is one of the most common microbes in the intestinal microbiota of healthy people and is believed to be a biomarker of intestinal health. A reduced abundance of Faecalibacterium is associated with disorders including inflammatory bowel disease and colorectal cancer. In patients treated with the solution of the present invention, an increase in Faecalibacterium was detected.

[0202] The administration of molecular hydrogen in the presence of salts according to the present invention has been shown to be safe in human beings (no adverse events reported) and produced a change in the intestinal microbiota detectable in the increase of Firmicutes and Faecalibacterium in the stool, and in the concomitant reduction of Proteobacteria (Figure 3). This activity is fundamental for correct intestinal homeostasis and is conducive to human well-being and health.

Claims

CLAIMS1 . An aqueous solution comprising molecular hydrogen, at least one salt, and ascorbic acid or a salt thereof, for use in reducing side effects associated with anti-cancer therapies, in particular chemotherapy and radiotherapy drugs, wherein said aqueous solution is administered percutaneously or transdermally.

2. The solution for use according to claim 1 , wherein molecular hydrogen is present in the solution in an amount (concentration) comprised in the range from 0.3 ppm (mg / L) to 3.6 ppm (mg / L), preferably from 0.4 ppm (mg / L) to 1 .2 ppm (mg / L), more preferably from 0.5 ppm (mg / L) to 0.7 ppm (mg / L), even more preferably of about 0.6 ppm (mg / L).

3. The solution for use according to claim 1 or 2, wherein the at least one salt is selected from the group consisting of NaCI (sodium chloride); NaHCOs (sodium bicarbonate); Na2[CO3] (sodium carbonate); NaaCeHsO? (sodium citrate); NaHPCh (sodium dihydrogen phosphate); CH2(COONa)2 (disodium malonate); KCI (potassium chloride); KHCO3 (potassium bicarbonate); K2[CO3] (potassium carbonate); K3C6H5O7 (potassium citrate KHPO3; K2PO3 (potassium hydrogen phosphate); KOCOCH(OH)CH(OH)COOH (potassium tartrate monobasic); KOCOCH(OH)CH(OH)COK (potassium tartrate); KOCOCH(OH)CH(OH)COONa (sodium potassium tartrate); MgCk (magnesium chloride); MgO (magnesium oxide); Mg(OH)2 (magnesium hydroxide); Mg[HCO3]2 (magnesium bicarbonate); MgCCh (magnesium carbonate); MgsfCsHsOzk (magnesium citrate); MgCioHi2N20e (magnesium pidolate); MgSCU (magnesium sulfate); Mg(HSO4)2 (magnesium hydrogen sulfate); CaCfe (calcium chloride); Ca[HCOs]2 (calcium bicarbonate); CaCOs (calcium carbonate); CastCeHsO / k (calcium citrate); CaHPO4 (calcium hydrogen phosphate); Ca(H2PO4)2 (calcium dihydrogen phosphate) and mixtures thereof.

4. The solution for use according to any one of claims 1 to 3, wherein said at least one salt is selected from hydrogencarbonate (bicarbonate) of an alkali metal or alkaline earth metal and chloride of an alkali metal or alkaline earth metal.

5. The solution for use according to any one of claims 1 to 4, wherein said at least one salt is hydrogencarbonate (bicarbonate) of an alkali metal or chloride of an alkali metal.

6. The solution for use according to any one of claims 1 to 3, wherein the at least one salt is selected from the group consisting of sodium hydrogencarbonate (bicarbonate), potassium hydrogencarbonate (bicarbonate) and magnesium chloride, and mixtures thereof.

7. The solution for use according to any one of claims 1 to 6, wherein the at least one salt is present in the aqueous solution in an amount in the range from 10 mM to 500 mM, preferably from 50 mM to 350 mM.

8. The solution for use according to any one of claims 1 to 3, 7, wherein said aqueous solution comprises at least two salts.

9. The solution for use according to claim 8, wherein said at least two salts are hydrogencarbonate (bicarbonate) of an alkali or alkaline earth metal and chloride of an alkali or alkaline earth metal, preferably of an alkali metal, even more preferably they are hydrogencarbonate (bicarbonate) of an alkali metal and magnesium chloride.

10. The solution for use according to claim 9, wherein said at least two salts are sodium or potassium bicarbonate and magnesium chloride.

11. The solution for use according to any one of claims 1 to 10, wherein the ascorbic acid is present in an amount comprised in the range from 10 mM to 500 mM, preferably from 50 mM to 350 mM.

12. The solution for use according to any one of claims 1 to 11 , wherein the aqueous solution of the invention has an ionic strength in the range from 0.098 mol / L to 0.25 mol / L, preferably from 0.2 mol / L to 0.24 mol / L, more preferably of about 0.220 mol / L.

13. The solution for use according to any one of claims 1 to 3, said solution comprising:- sodium bicarbonate, potassium bicarbonate, magnesium chloride and ascorbic acid, in a weight ratio of 1 :1 :1 :1 (solution A);- sodium bicarbonate, potassium bicarbonate, magnesium chloride and ascorbic acid, in a weight ratio of 2:2:2:2 (solution B);- sodium bicarbonate, potassium bicarbonate, magnesium chloride and ascorbic acid, in a weight ratio of 3: 1:3:2 (solution E);- sodium bicarbonate, potassium bicarbonate, magnesium chloride and ascorbic acid, in a weight ratio of 1:2:3: 1 (solution F);- sodium bicarbonate, potassium bicarbonate, magnesium chloride and ascorbic acid, in a weight ratio of 1 :2:3:3 (solution G);- sodium bicarbonate, potassium bicarbonate, magnesium chloride and ascorbic acid, in a weight ratio of 2: 1:3:2 (solution H);- sodium bicarbonate, potassium bicarbonate, magnesium chloride and ascorbic acid, in a weight ratio of 0:1 :2:1 (solution I);- sodium bicarbonate, potassium bicarbonate, magnesium chloride and ascorbic acid, in a weight ratio of 0:3:4:2 (solution J);- sodium bicarbonate, potassium bicarbonate, magnesium chloride and ascorbic acid, in a weight ratio of 3:0:3: 1 (solution K);- sodium bicarbonate, potassium bicarbonate, magnesium chloride and ascorbic acid, in a weight ratio of 0: 1:3:3 (solution L);- sodium bicarbonate, potassium bicarbonate, magnesium chloride and ascorbic acid, in a weight ratio of 2:2:3:2 (solution M), and- sodium bicarbonate, potassium bicarbonate, magnesium chloride and ascorbic acid, in a weight ratio of 0:3:3: 1 (solution N).

14. The solution for use according to any one of claims 1 to 13, wherein the side effects of anti-cancer therapies include at least one from reduction of nausea, headache, hair loss, anemia, decreased immune defenses, vomiting, diarrhea, inflammation or infection of the mouth, insomnia, asthenia, fibromyalgia, excessive weight loss, liver damage, cognitive problems (chemo brain) and musculoskeletal pain.

15. An aqueous solution comprising molecular hydrogen, at least one salt, and ascorbic acid or a salt thereof, for use in reducing inflammatory processes generated by a disease selected from the group consisting of arthritis, arthrosis, rheumatoid arthritis, fatigue, neuropathy even induced by drugs, celiac disease, psoriasis tendonitis, tennis elbow, pubalgia, muscle strain and sprain, wherein said aqueous solution is administered percutaneously or transdermally.

16. An aqueous solution comprising molecular hydrogen, at least one salt, and ascorbic acid or a salt thereof for use in the treatment of intestinal dysbiosis, wherein said aqueous solution is administered percutaneously or transdermally.

17. Cosmetic use of an aqueous solution comprising molecular hydrogen, at least one salt, and ascorbic acid or a salt thereof, in the positive modulation of the intestinal microbiota, wherein said aqueous solution is administered percutaneously or transdermally.

18. An aqueous solution comprising molecular hydrogen, sodium hydrogencarbonate, potassium hydrogencarbonate, magnesium chloride, and ascorbic acid or a salt thereof, wherein the weight ratio of sodium hydrogencarbonate, potassium hydrogencarbonate, magnesium chloride, and ascorbic acid or a salt thereof is in the range of 0:0:1 :1 to 3:3:3:3 provided that- at least one of sodium hydrogencarbonate and potassium hydrogencarbonate is presentand wherein when ascorbic acid is present, the solution comprises- sodium bicarbonate, potassium hydrogencarbonate, magnesium chloride and ascorbic acid in a weight ratio of 0:1 :3:3 (solution L), or - sodium hydrogencarbonate, potassium hydrogencarbonate, magnesium chloride and ascorbic acid in a weight ratio of 2:2:3:2 (solution M), or- sodium hydrogencarbonate, potassium hydrogencarbonate, magnesium chloride and ascorbic acid in a weight ratio of 0:3:3:1 (solution N).

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