COMBINATION OF MICRONUTRIENTS TO STIMULATE THE ENDOGENOUS PRODUCTION OF HYDROGEN SULFIDE (H2S)

Abstract

The present invention relates to pharmaceutical, dietary and/or food compositions exerting the ability to activate the endogenous production of hydrogen sulfide (H.sub.2S) so to feed the redox buffer capacity and to boost the natural ability to compensate both oxidative and reductive stress.

The invention further relates to the use of the aforementioned compositions to correct signs of metabolic derangements such as: oxy-redox imbalances including reductive stress, mitochondrial dysfunction and bioenergetics, insulin resistance, endothelial dysfunction and hyperhomocysteinemia. Accordingly, the composition of the present invention can be used as a medicament for the treatment of hypertension, atherosclerosis and cardiovascular disease, erectile dysfunction, viral infections and inflammatory lung injuries, diabetes and PCOS, autoimmune diseases, neurodegeneration, repeated implantation failure and repeated miscarriage and infertility in both men and women.

Claims

1. A combination comprising: a) a cysteine donor; b) taurine and/or the pharmaceutically acceptable salts thereof; c) vitamin B6 and/or the pharmaceutically acceptable salts or derivatives thereof.

2. The combination according to claim 1, wherein the cysteine donor is in amounts ranging from 40 to 60% by weight, taurine is in amounts ranging from 40 to 60% by weight, vitamin B6 is preferably in amounts ranging from 0.1 to 5% by weight, with respect to the sum of components a), b) and c).

3. The combination according to claim 1, wherein the cysteine donor is L-cystine or N-acetylcysteine.

4. The combination according to claim 1, wherein vitamin B6 is in the form of pyridoxine or pyridoxine hydrochloride or pyridoxal-5-phosphate.

5. A composition comprising the combination according to claim 1 and at least one physiologically acceptable excipient.

6. The composition according to claim 5, wherein the physiologically acceptable excipient is selected from the group consisting of bulking agents, anticaking agents, stabilizers and a mixture thereof.

7. The composition according to claim characterized in being in the form of tablet, capsule, granule, soluble granule, drinkable solution, lavage solution, breathable solution, injection or suppository.

8. (canceled)

9. A method for decreasing circulating homocysteine in a subject, comprising the step of administering to the subject separately, simultaneously or sequentially the combination of claim 1, wherein the subject has a wild type genotype or carries a defective gene for the enzymes MTHFR, MTR, MTRR, CHDH, CBS, or any combination thereof.

10. The method according to claim 9, wherein the cysteine donor is administered at a dosage comprised between 100 and 2000 mg per day.

11. The method according to claim 9, wherein taurine is administered at a dosage between 100 and 2000 mg per day.

12. The method according to claim 9, wherein pyridoxal-5-phosphate is administered at a dosage between 1 and 100 mg per day.

13. (canceled)

14. The method according to claim 9, for the correction of insulin resistance or for the treatment of diabetes or PCOS.

15. The method according to claim 9, for the treatment and prevention of myocardial infarction, reperfusion injury, hearth failure, atherosclerosis, or erectile dysfunction.

16. The method according to claim 9, for the treatment of hypertension.

17. The method according to claim 9, for the prevention and/or treatment of viral infections.

18. The method according to claim 17, wherein such viral infection is Coronavirus.

19. The method according to claim 9, for the treatment of neurodegenerative diseases.

20. The method according to claim 9, to improve immune tolerance in donor transplant recipients, whether or not suffering from host versus graft disease, or for the treatment of autoimmune diseases.

21. The method according to claim 9, for the support to immune tolerance and placentation in pregnancy, or for the treatment of recurrent implantation failure, recurrent miscarriage, or premature labor.

22. The method according to claim 9, for the treatment of couple's infertility both in natural conception and within ART cycles, or for the treatment of isolated idiopathic female infertility, idiopathic male infertility, or male infertility consequent to varicocele.

23. The method according to claim 9, for improving the oxy-redox balance, the epigenetic processes, or the energy output of ovarian oocytes/follicles, or embryos and stem cells undergoing in vitro culture.

24. The method according to claim 9, wherein the subject is a mammal.

Description

EXAMPLES

Example 1

[0120] A tablet for oral administration was formulated by including all the targeted nutritional substances as follows: L-Cystine 125 mg; taurine 125 mg; pyridoxal-5-phosphate 5 mg. The excipients included calcium phosphate, magnesium salt of fatty acids, cross-linked carboxymethyl cellulose, and silicon dioxide.

[0121] The resulting tablet had a global mass of 520 mg and was of white color, odorless and tasteless. The stability of the tablet was investigated under standard (25° C.±2° C.; 60% R.H.±5%) conditions up to 36 months and under accelerated conditions (40° C.±2° C.; 75% R.H.±5%) up to 9 months. The stability was assessed by monitoring the organoleptic properties, by checking the microbial load and by titration of the nutritional substances. All the results were within the approved range up to the end of the stability program in both the tested conditions.

Example 2

[0122] Ten healthy subjects (5 women) aged between 30 and 45 years, non-smokers and with borderline hyperhomocysteinemia assumed the composition formulated according to EXAMPLE 1, 2 tablets in the morning and 2 tablets in the evening. A sample of venous blood was taken in the morning within 9.00 hours after overnight fasting at baseline and after one week of treatment. The metabolites were detected by mass spectrometry or ion chromatography as appropriate. The average homocysteine concentration dropped from 13.8 to 8.3 μMol/L with a 40% reduction. Noteworthy, homocysteine decreased in all subjects, including those starting with a normal value. There were little changes of cysteine (+6%) and GSH (+7%) concentration and of the GSH to GSSG ratio (+14%). In contrast, there was a sharp increase of lanthionine (+83%), indicating increased H.sub.2S generation from CBS, of homolanthionine (+64%), indicating increased H.sub.2S generation from CSE, and of 3-mercaptopyruvate (+54%) and sulfate (+41%), indicating increased H.sub.2S generation from 3MST and increased H.sub.2S oxidation in mitochondria for the generation of ATP. Thus, the combination was very effective in reducing homocysteinemia and the effect was associated to a sharp increase of the release of H.sub.2S and to ATP generation.

[0123] Advantageously, the regular assumption of the composition according to the invention may help to restore adequate H.sub.2S release and to reduce blood fasting homocysteine.

[0124] Advantageously, the regular assumption of the composition according to the invention may help to correct mitochondrial dysfunction.

Example 3

[0125] Five male subjects aged 30-40, following a healthy diet and virtually free from diseases were tested for plasma NADH concentration by enzymatic cycling reaction and for erythrocyte GSH after assuming vitamin C, 2 gr per day during 1 week and, after a 2 week wash out, vitamin C, 2 gr per day, together with the composition formulated according to EXAMPLE 1, 4 tablets per day, for 1 week. Vitamin C caused a subclinical reductive stress as reported by a 40% increase of plasma NADH and 31% and 63% increase of erythrocyte GSH and GSH to GSSG ratio, respectively. The add-on of the composition formulated according to EXAMPLE 1 completely prevented these changes with just 7% increase of NADH, and an as well very small increase of GSH (8%) and GSH to GSSG ratio (17%). Thus, the aforementioned composition prevented the reductive stress induced by a powerful reducing agent.

[0126] Advantageously, the composition according to the invention positively modifies the oxy-redox balance and prevents the reductive stress induced by powerful reducing substances.

Example 4

[0127] Eight patients (4 women) aged between 40 and 60 years with newly diagnosed insulin resistance were sampled at baseline and after 3 months of treatment with the composition formulated according to EXAMPLE 1, 2 tablets in the morning and 2 tablets in the evening. Insulin resistance estimated as HOMA-IR decreased from 3.1 to 2.5 and glycated hemoglobin (HbA1c) from 7.1 to 6.2, both indicating an improved glucose homeostasis. Malondialdehyde (MDA) was tested by thiobarbituric acid reactive substance assay as an index of lipid oxidative damage and decreased from 328 to 217 indicating a decreased oxidative load. Thus, the composition according to the invention was effective in reducing insulin resistance.

[0128] Advantageously, the composition according to the invention decreases the oxidative load allowing the correction of insulin resistance.

Example 5

[0129] Bacterial lipopolysaccharide (LPS) is known to induce endothelial dysfunction. Ten adult rabbits were induced endotoxic shock by a single LPS bolus (Escherichia coli endotoxin, 0.5 mg/kg iv). Five of the rabbits were pre-treated for 2 weeks by oral gavage of a solution containing the composition formulated according to EXAMPLE 1: 4 tabs dissolved in 100 ml water, daily oral gavage with 3 ml of the achieved solution. One day after LPS injection, untreated rabbits showed increased circulating polynuclear neutrophils and fibrinogen with reduced platelet count, coagulation factors II and V and Protrombin index. The same changes occurred in pre-treated rabbits at a far lower extent indicating a prevention of the coagulopathy from LPS. Two out of 5 non pre-treated rabbits died before day 5 after LPS whereas all pre-treated animals survived up to day 5 post LPS when the animals were sacrificed and aorta segments were collected to check endothelial damage. Endothelial injury was present on 31% of the tested area of control rats whereas injuries did not exceed 5% in pre-treated animals.

[0130] Advantageously, the composition according to the invention prevents the endothelial dysfunction induced by bacterial LPS.

Example 6

[0131] Twenty male subjects with newly diagnosed arterial hypertension and with a diastolic blood pressure >90 mmHg and a systolic blood pressure >130 mmHG assumed the composition formulated according to EXAMPLE 1, 4 tablets per day, for 2 weeks. Blood pressure was measured by mercury sphygmomanometer according to the standard clinical practice at baseline, at the end of the 2-week treatment and after a further week of wash-out. After treatment, the average diastolic blood pressure decreased from 93.25 to 87.8 mm Hg (−5.45) and the average systolic blood pressure decreased from 134.05 to 125.75 mm Hg (−8.3). Diastolic and systolic blood pressure returned to baseline values (respectively 92.75 and 132.85 mmHg) after one-week wash-out.

[0132] Advantageously, the composition according to the invention decreases blood pressure in hypertensive subjects.

Example 7

[0133] Twenty Balb/c mice were intranasally infected with 10.sup.7 pfu of Respiratory Syncytial Virus (RSV) grown in and purified from Hep-2 cells. Ten mice were pre-treated for 1-week pre-infection and 1-week post-infection by oral gavage of a solution containing the composition formulated according to EXAMPLE 1: 2 tabs dissolved in 50 ml water, daily oral gavage with 1 ml of the achieved solution. The other ten infected mice served as controls. The loss of body weight following infection and the viral-induced illness score were recorded. On day 3 post-infection the weight loss in treated animals was 8.4% compared to 18.8% in untreated animals The average illness score was, respectively for treated and untreated animals 1.1 vs 2.3 at day 1; 1.3 vs 2.95 at day 2; 0.25 vs 3.3 at day 3; 0 vs 0.9 at day 4. All animals were symptom free at day 5. Thus, the treatment was effective in reducing the burden of RSV infection in mice.

[0134] Advantageously, the composition according to the invention can be used to treat respiratory viral infections.

Example 8

[0135] An animal model of Ventilation Induced Lung Injury (VILI) was established by exposing 10 anesthetized rats to 4-hour mechanical ventilation at tidal volume of 30 mL/kg, respiratory rate of 50/min, inspiratory/expiratory ratio of 1:1, and FiO2 of 50%. Five of the rats were pre-treated during one week by oral gavage of a solution containing the composition formulated according to EXAMPLE 1: 4 tabs dissolved in 100 ml water, daily oral gavage with 1 ml of the achieved solution. The animals were sacrificed at the end of the exposure to calculate the lung injury score (from 0, no injury, to 4, more than 75% injured) and the edema rate by the lung wet-to-dry ratio. Untreated animals had a lung injury score of 3.75 and a lung wet-to-dry ratio of 7.5 compared to, respectively, 1.9 and 3.8 in pre-treated animals indicating that the treatment had prevented the lung injury to a large extent.

[0136] Advantageously, the composition according to the invention can be used to prevent VILI.

Example 9

[0137] The transgenic 3×Tg-AD mice overexpress human amyloid precursor protein (APP), PS1 and tau protein and develop severe Alzheimer's disease. Ten, 6-month old 3×Tg-AD mice were treated during 12 weeks (up to the age of 9 months) by oral daily gavage of a solution containing the composition formulated according to EXAMPLE 1: 2 tabs dissolved in 50 ml water, daily oral gavage with 1 ml of the achieved solution. Other 10, 6-month old mice did not receive the treatment and served as controls. By the age of 9 months treated mice, as compared to untreated animals, showed improved cognitive function with respect to the Morris water maze test (escape latency, target zone and target crosses). One animal from each group was sacrificed and target cortex areas were analyzed to calculate the amyloid beta plaque extent: the untreated animal showed extensive amyloid deposition, which was barely detectable in the treated mouse.

[0138] Advantageously, the composition according to the invention exerts its activity also within the central nervous system. Advantageously, the composition according to the invention may be used for the treatment and prevention of neurodegenerative diseases linked to lack of H.sub.2S including, but not limited to, Alzheimer's disease and Parkinson's disease.

Example 10

[0139] Five patients suffering from psoriasis vulgaris at progressive stage assumed the composition formulated according to EXAMPLE 1, 4 tablets per day, for 2 weeks. At the end of the treatment a sample of blood was taken from these patients and from other 5 age and disease stage matched psoriasis patients to isolate Treg and Tresp lymphocytes. Isolated cells were tested for both the Treg ability to suppress the Tresp proliferation (as triggered by IL-2 and anti-CD28) and the ability to proliferate of Tregs (CSFE labelling). More than 50% of the Tresp from untreated patients proliferated in spite of the expected Treg inhibition whereas only 32% of the Tresp from treated patients proliferated. Opposite, about 45% of the Tregs from treated patients was able to proliferate of compared to less than 30% of those from untreated patients.

[0140] Advantageously, the composition according to the invention exerts the ability to boost Treg lymphocyte function in humans. Advantageously, the composition according to the invention can be used for the treatment of autoimmune diseases.

Example 11

[0141] C57BL/6 HO-1 knock-out mice do not express HO-1 and are unable to generate alive offspring due to lack of placentation. Female and male C57BL/6 HO-1 heterozygote knock-out mice (HO-1.sup.−/+) were cross-breed and their offspring was investigated. The females of 10 mice couples were treated during 2 weeks before mating by oral daily gavage of a solution containing the composition formulated according to EXAMPLE 1: 2 tabs dissolved in 50 ml water, daily oral gavage with 1 ml of the achieved solution. Their offspring was compared to that from other 10 untreated mice couples. Pre-treated couples produced an average of 6.5 pups/litter, whereas untreated couples produce just 4.1 pups/litter. Moreover, the birth weight of pups from untreated mice couple was about 30% lower than that from treated couples. In summary, the treatment prevented the low reproductive efficiency and placentation of genetically weak animals and is expected to do the same in human pregnancies.

[0142] Advantageously, the composition according to the invention exerts the ability to resume low efficient placentation from lack of HO-1/CO/H.sub.2S activation. Advantageously, the composition according to the invention can be used for the treatment of recurrent implantation failure and recurrent miscarriage.

Example 12

[0143] Couples referring to an assisted reproduction clinic and put on the waiting list were offered a nutritional supplementation for both partners with the composition formulated according to EXAMPLE 1, 4 tablets per day, as a preparation for the treatment. Matched couples refusing the supplementation served as controls. A total of 24 couples started the supplementation and were observed for an average of 3.2 months whereas 21 couples serving as controls were followed up for an average of 2.9 months. Nine out of 24 treated couples (37.5%) experienced a spontaneous pregnancy during the waiting time compared to 1 out of 21 (5%) in the control group.

[0144] Advantageously, the composition according to the invention can be used for the treatment of couple's infertility.

Example 13

[0145] Ten couples with more than 2 previous Assisted Reproductive Technologies (ART) failures were offered a nutritional supplementation for both partners with the composition formulated according to EXAMPLE 1, 4 tablets per day, during 3 months in preparation of a new ART cycle and thereafter during the hormonal stimulation. Other ten couples with the same characteristics and refusing the supplementation were observed for a similar period before a new ART attempt and served as controls. After the new ART cycle, 7 out of 10 pre-treated couples achieved a clinical pregnancy compared to 2 of 10 couples in the control group.

[0146] Advantageously, the composition according to the invention can be used for the treatment of couple's infertility within ART programs.

Example 14

[0147] Surgical varicocele was induced in 20 adult male Wistar rats. Two months post-surgery a significant damage to testis and sperm cells was confirmed and 10 of the varicocele rats received a treatment with the composition formulated according to EXAMPLE 1, 4 tabs dissolved in 100 ml water, daily oral gavage with 1 ml of the achieved solution, the other 10 rats served as controls. Two months later, i.e. 4 months after surgery, untreated rats had oligospermia, asthenospermia and increased lipid peroxidation whereas treated rats had only minor decrease of sperm number and quality and lipid peroxidation had returned close to baseline. Six months later, i.e. 8 months after surgery, the rat testes of untreated rats showed intensive iron deposition in germinal cells and interstitial space indicating a defective activity of HO-1 with release of ferric iron within the tissues. Due to the oxidative damage caused by iron accumulation the spermatogenesis was unlikely to be restored in these animals. Treated rats presented only very minor iron staining in parallel with their preserved spermatogenesis.

[0148] Advantageously, the composition according to the invention prevents HO-1 inactivation and testicular iron deposition in varicocele. Advantageously, the composition according to the invention can be used to treat human varicocele.

Example 15

[0149] Bovine cumulus-oocyte complexes (COC) were obtained from the ovaries of slaughtered cows. COCs were aspirated from antral follicles (3-8 mm in diameter) and oocytes with at least four layers of cumulus cells with homogenous cytoplasm were selected. A sample of COCs was used to check the nuclear maturation stage at time of sampling (n=20). Control COCs (Group 1) were matured in vitro using 500 μL of TCM199 medium (Gibco; Invitrogen Co., Grand Island, N.Y., USA) supplemented with FSH and hCG and with 10% (v:v) calf serum (N=20). Sibling bovine COC (n=20) were cultured with the same medium further supplemented with 100 μL of a solution containing the composition formulated according to EXAMPLE 1 where the amount of substances equivalent to a daily human dose had been diluted in 1 L (Group 2). Out of 20 COCs tested at time of sampling, 15 were at GV stage, 5 at intermediate stage and none was at MII stage. The development of the oocytes was assessed after 24 hours of in vitro culture: Group 1 produced 4 GV, 5 intermediate and 11 MII stage (matured) oocytes; Group 2 produced 2 GV, 3 intermediate and 15 MII oocytes (+20% vs Group 1) indicating that the addition of the composition formulated according to EXAMPLE 1 had improved the maturation rate. After in vitro fertilization of the MII oocytes obtained from the previous experiment blastocyst formation rate was 4/11 (36%) in Group 1, which improved to 9/15 (60%) in Group 2.

[0150] Advantageously, the in vitro supplementation of the culture medium with a solution containing the composition listed in EXAMPLE 1 results in an increased rate of in vitro maturation of ex-vivo oocytes and in a higher rate of blastocyst development. The composition according to the present invention may improve the efficiency of the in vitro culture of oocytes, embryos and stem cells.