[0001] Numerous functions of reduced glutathione (GSH), atripeptide consisting of glycine, glutamic, acid and cysteine, have been established. (Hammond CL, Lee TK, Ballatori N. Novel roles for glutathione in gene expression, cell death, and membrane transport of organic solutes. J Hepatol. 2001;34:946-54.)* They can be broadly classified in two types: as an antioxidant and as a key molecule in detoxification of endo- and xenobiotics.

[0002] GSH is the most abundant intracellular non-protein thiol and as such is critical for the maintenance of the intracellular redox homeostasis. (DeLeve LD,.Kaplowitz N. Glutathione metabolism and its role in hepatotoxicity. Pharmacol Ther. 1991;52:287-305.)* Cells, and particularly their mitochondria, are continuously exposed to reactive oxygen species (ROS). Mitochondrial glutathione (mGSH) is the main line of defense for the maintenance of the appropriate mitochondrial redox environment to avoid or repair oxidative modifications leading to mitochondrial dysfunction and cell death. (O'Donovan Dl.Fernandes CJ. Mitochondrial glutathione and oxidative stress: implications for pulmonary oxygen toxicity in premature infants. Mol. Genet. Metab 2000;71 :352-8.)*

[0003] Mitochondria are the primary intracellular sites of oxygen consumption during exercise and the major source of reactive oxygen species (ROS), most of them produced from the mitochondrial respiratory chain. The ratio between reduced and oxidized glutathione (GSH / GSSG) can act as a marker of antioxidant status in various pathological and physiological conditions, including exercise. (Oncol Lett. 2012 December; 4(6): 1247- 1253. Published online 2012 September 21. doi: 10.3892/ol.2012.931).)*

[0004] Physical exercise decreases the reduced form and increases the oxidized form of glutathione (D.E. Laaksonen, M. Atalay, L. Niskanen, M. Uusitupa, O. Hanninen, C.K. Sen Blood glutathione homeostasis as a determinant of resting and exercise-induced oxidative stress in young men Redox Rep., 4 (1-2) (1999), pp. 53-59; J Appl Physiol (1985). 1988 Jan;64(l): 115-9. Blood glutathione oxidation during human exercise.)* and prolonged exercise decreases total plasma and tissue glutathione content over time. (P.M. Tiidus, J. Pushkarenko, M.E. Houston. Lack of antioxidant adaptation to short-term aerobic training in human muscle Am. J. Physiol., 271 (1996), pp. R832-R836.)* Additionally, it has been shown that supplementation with GSH can suppress muscle fatigue in humans. (Aoi et al. Journal of the International Society of Sports Nutrition (2015) 12:7 DOI 10.1186/s 12970- 015-0067-x. Glutathione supplementation suppresses muscle fatigue induced by prolonged exercise via improved aerobic metabolism.)* Gohil et al (Gohil Kl, Viguie C, Stanley WC, Brooks GA, Packer L.)* show a decrease of 60% of blood GSH from control and 100% increase in blood GSSH (oxidized form) from control after prolonged submaximal test for 90 minutes at 65% V02max. (See also Chandan K. Sen, Glutathione homeostasis in response to exercise training and nutritional supplements, Molecular and Cellular Biochemistry 196: 31-42, 1999.

[0005] GSH also plays a role in the metabolization of lactic acid produced during exercise. During muscle contraction, lactic acid, a major source of protons, is rapidly produced by increased glycolytic metabolism, lowering the pH and inhibiting muscle contraction. Animal and human studies indicate that long term supplementation using high doses of GSH can inhibit the decrease in intermuscular pH after exercise.

[0006] However, it is unknown the impact on metabolism that the supplementation of GSH during acute GSH depletion produced by intense exercise. All previous art have used oral GSH (high doses, long term) which has been proved to have a very poor bioavailability and thus are unsuitable for fast delivery.*

[0007] GSH has also a critical role in the immune system. Moderate changes in the intracellular GSH levels may have profound effects on lymphocytes functions. Certain functions, such as the DNA synthesis, are very sensitive to reactive oxygen intermediates and, therefore, are favored by high levels of GSH. Optimal GSH levels in lymphocytes are a good marker for health. (Proc Nutr Soc. 2000 Nov; 59(4):595-600. Glutathione and immune function.)*

[0008] In detoxification pathways, GSH (a nucleophile) reacts with electrophilic metabolites of endo- and xenobiotics to form GSH adducts. These are excreted after further metabolism to form the corresponding cysteine adduct, in many cases, the N-acetylcysteine adduct.

[0009] The conjugation reaction can occur spontaneously but is often catalyzed by one of the glutathione S-transferases. These enzymes markedly speed up the conjugation of electrophiles with GSH and thereby prevent the reaction of potentially toxic metabolites with vitally important macromolecules. (Hammond CL, Lee TK, Ballatori N. Novel roles for glutathione in gene expression, cell death, and membrane transport of organic solutes. J Hepatol. 2001;34:946-54.)*

[0010] One example of this mechanism of action is the conjugation of GSH with the hepatotoxic metabolite of paracetamol: N-acetyl-p-benzoquinoneimine (NAPQI). When intracellular GSH is depleted, this metabolite is able to react with critical macromolecules and a process which ultimately leads to necrosis and apoptosis is set in motion. (LAURA P. JAMES, PHILIP R. MAYEUX, AND JACK A. HINSON. ACETAMINOPHEN-INDUCED HEPATOTOXICITY Departments of Pediatrics (L.P.J.) and Pharmacology and Toxicology (L.P.J., P.R.M., J.A.H.), University of Arkansas for Medical Sciences, Little Rock, Arkansas.)*

[0011] GSH plays an important role in the detoxification of ethanol and acute ethanol administration leads to GSH depletion in the liver and other tissues. Hepatic GSH levels have been shown to be depleted after acute ethanol administration in animals. When GSH levels are enhanced by administration of GSH or its precursors, the depletion of GSH levels by ethanol is prevented. (Richie JP Jr. Glutathione depletion and recovery after acute ethanol administration in the aging mouse Biochem Pharmacol. 2007 May 15;73(10):1613-21. Epub 2007 Jan 30.)*

[0012] However, it is unknown the impact of GSH supplementation during acute GSH depletion produced by alcohol ingestion may have in the metabolism. The previous art has been focused on long term effects of GSH supplementation in alcoholic chronic depleted subjects. High doses of GSH solutions in injectable form have been tested, but the effect of low doses of GSH supplementation in healthy subjects during acute depletion is unknown.

[0013] GSH is produced naturally by the human body. A healthy adult human liver alone produces about 14 grams per day of GSH, replacing the natural depletion of GSH in the body that results from the functions of normal daily living. During activities and conditions that result in acute depletion of GSH in the body, GSH depletion in the body is hastened. This might also be described as an acute depletion of GSH. Although the synthesis of GSH in the body is stimulated when its concentration decreases, the instant availability of precursor amino acids, particularly the availability of cysteine, is not sufficient to sustain intracellular levels of GSH. Therefore, during acute GSH depletion there is a window of up to several hours until the level of GSH returns to baseline. During this period, damage produced by ROS can occur and an efficient supplementation of GSH can have a traceable impact.

[0014] As a pharmaceutical drug, GSH has been used as injectable form, for therapies related to ethyl alcohol or drug intoxication (anti-neoplastic chemotherapy, anti-tuberculosis, neuroleptics, anti-depression and paracetamol, acting as antidote for poison). It also may assist in prophylaxis and treatment of the damages from ionizing radiation.

[0015] Further, the impact of long term GSH supplementation (orally, intravenous, etc.) is limited unless the subject is continuously depleted, as in the case of chronic patients, or supplementation is done using a large dose (grams per day) for a very long period (months).

[0016] On the other hand, during situations of acute depletion of GSH the effect of GSH supplementation can be easily detected, as in the case of injections of GSH for ameliorating the effects of drug intoxication.

[0017] Healthy individuals are exposed frequently to events of acute depletion of GSH in their daily life. Most of the acute GSH depletion in healthy individuals happens during intense exercise, alcohol drinking, mental stress and other physical stress (like seasonal colds).

[0018] Healthy individuals could benefit from GSH supplementation during acute GSH depletion, as it would probably help reducing the effects produced by the depletion. However, GSH in its injectable form is not an option for healthy individuals and oral supplementation of GSH, mainly in form of capsules, powder, liquid pills, etc., has limited effectiveness due to their very poor bioavailability. Orally administered and ingested GSH is successively degraded to y-glutamyl-X (where X is another aminoacid) and cystenyl-glycine. Cystenyl-glycine is degraded into cysteine and glycine by peptidases. (Park EY1, Shimura N, Konishi T, Sauchi Y, Wada S, Aoi W, Nakamura Y, Sato K. Increase in the protein-bound form of glutathione in human blood after the oral administration of glutathione, J Agric Food Chem. 2014 Jul 2;62(26):6183-9. doi: 10.1021/jf501338z. Epub 2014 Jun 11; Witschi, A, Reddy, S., Stofer, B., Lauterburg, B. H. The systemic availability of oral glutathione. Eur. J. Clin. Pharmacol. 1992,43, 667-669.)*

[0019] Moreover, oral supplementation has been shown to have an almost null effect on GSH levels in the first 2 hours after ingestion. (Park EY1 , Shimura N, Konishi T, Sauchi Y, Wada S, Aoi W, Nakamura Y, Sato K. Increase in the protein-bound form of glutathione in human blood after the oral administration of glutathione. J Agric Food Chem. 2014 Jul 2;62(26):6183-9. doi: 10.1021/jf501338z. Epub 2014 Jun 11.)* The short time frame during acute depletion makes oral GSH an unsuitable option for GSH supplementation during acute GSH depletion.

[0020] Chewing gum has been proved to be an excellent delivery system for drugs and nutrients, increasing bioavailability and the rate at which nutrients and other molecules are absorbed by the human body. Examples are nicotine, caffeine and vitamin loaded chewing gum. (Kamimori GH1, Karyekar CS, Otterstetter R, Cox DS, Balkin TJ, Belenky GL, Eddington D.Int The rate of absorption and relative bioavailability of caffeine administered in chewing gum versus capsules to normal healthy volunteers. J Pharm. 2002 Mar 2;234(1- 2):159-67; Prashant K. Pagare, Chandrakant S. Satpute, Varsha M. Jadhav and Vilasrao Kadam Medicated Chewing Gum: A Novel Drug Delivery System Journal of Applied Pharmaceutical Science 02 (06); 2012: 40-54.)*

[0021] As explained, the previous art describes either long term, high dosage effects of oral supplementation and or short term, high dosage effects of GSH injections in unhealthy subjects. It is unknown the impact of GSH supplementation during acute GSH depletion in healthy subjects.

Summary of the Invention

[0022] The present invention provides an effective and fast delivery system and its use by healthy individuals during situations and conditions of acute GSH depletion in the body. The present invention provides a chewable delivery system for GSH in the form of a chewable base composition comprising a gum base and reduced glutathione.

[0023] The present invention further provides a method of reducing blood alcohol concentration in a human, comprising the steps of: i) providing one or more units of a chewable composition comprising a gum base and an amount of reduced glutathione; ii) providing a human who has consumed ethyl alcohol and has a blood alcohol level, or who will imminently consume ethyl alcohol and thereby induce a blood alcohol level; and iii) masticating by the human the one or more units of the chewable composition for a time sufficient to release a quantity of the reduced glutathione into the oral cavity of the human, for absorption into the blood; thereby reducing the blood alcohol concentration in the mammal. The method can also ameliorate the rate of alcohol metabolization in the mammal. The masticating of the units can begin after the human has consumed ethyl alcohol. The method can reduce the blood alcohol concentration in the mammal within 20 minutes, including within 10 minutes, and including within 5 minutes.

[0024] The present invention further provides a method of reducing a rise in blood alcohol concentration in a mammal following consumption of a quantity of ethyl alcohol, comprising the steps of: i) providing one or more units of a chewable composition comprising a gum base and an amount of reduced glutathione; and ii) masticating by the mammal the one or more units of the chewable composition for a time sufficient to release a quantity of reduced glutathione into the oral cavity of the mammal, for absorption into the blood, prior to or while consuming a quantity of ethyl alcohol that is sufficient to effect a rise in the blood alcohol concentration of a mammal; thereby reducing the rise in blood alcohol concentration in the mammal. The consumed quantity of ethyl alcohol is sufficient to effect a rise in the blood alcohol concentration of the mammal to at least 0.1 gm/L. The masticating of the one or more units of the chewable composition can be prior to the consuming of the quantity of ethyl alcohol.

[0025] The present invention further provides a method of reducing a perception of fatigue in a mammal during or after an intense exercise, comprising the steps of: i) providing one or more units of a chewable composition comprising a gum base and an amount of reduced glutathione; and ii) masticating by the mammal the one or more units of the chewable composition for a time sufficient to release a quantity of the reduced glutathione into the oral cavity of the mammal, for absorption into the blood, prior to or while performing an intense exercise that results in a perception of fatigue in the mammal that is associated with glutathione depletion in the blood of the mammal; thereby reducing the perception of fatigue in the mammal during or after the intense exercise, The masticating of the one or more units of the chewable composition can be prior to performing an intense exercise.

[0026] The present invention further provides a method of ameliorating a recovery from a perception of fatigue in a mammal during or after an intense exercise, comprising the steps of: i) providing one or more units of a chewable composition comprising a gum base and an amount of reduced glutathione; and ii) masticating by the mammal the one or more units of the chewable composition for a time sufficient to release a quantity of the reduced glutathione into the oral cavity of the mammal, for absorption into the blood, prior to or while performing an intense exercise that results in a perception of fatigue in the mammal that is associated with glutathione depletion in the blood of the mammal; thereby ameliorating the recovery from the perception of fatigue in the mammal during or after the intense exercise. The masticating of the one or more units of the chewable composition can be prior to performing an intense exercise.

[0027] The present invention provides a method of reducing a hastened depletion of glutathione or ameliorating the recovery of glutathione in a mammal, caused by an acute depleting event, comprising the steps of: i) providing one or more units of a chewable composition comprising a gum base and an amount of reduced glutathione; ii) providing a mammal that is experiencing, or who will imminently experience, an acute depleting event that hastens an acute depletion of glutathione in the mammal; and iii) masticating by the mammal the one or more units of the chewable composition for a time sufficient to release a quantity of the reduced glutathione into the oral cavity of the mammal, for absorption into the blood; thereby reducing the hastened depletion of, or ameliorating the recovery of, glutathione in the mammal, during or after the acute depleting event.

[0028] In a further aspect of the invention, a method further reduces the effects of the hastened depletion of glutathione caused by the acute depleting event, on the condition of the mammal.

[0029] The acute depleting event can be a condition or activity of the human or mammal that can extend up to 3 days, and more particularly up to 3 hours, in which the depletion of GSH in the body is caused by the acute depleting event at a rate or to an extent that exceeds the depletion rate or extent of the body during normal daily living. The acute depleting event can be the consumption of ethyl alcohol, intense exercise, stress, migraines, administration of a drug or medicament; administration of acetaminophen, an influence, exposure or consumption of a heavy metal, a bacterial or viral infection, an event that results in an accumulation of lactic acid in the blood, and an event that results in an increase in free radicals into the cells of the mammal. The method reduces the effects of the these acute depleting event on the mammal or human.

[0030] The present invention also provides a unit of a chewable composition, the unit comprising: a chewable base core comprising a gum base, a sweetener, and 0.5 - 15%, by weight of the chewable base core, an active ingredient consisting of reduced glutathione; and a coating surrounding the chewable base core, having a harder structure than the chewable base core, and comprising a sweetener. The active ingredient can be 1.0 - 5.0 %, by weight of the chewable base core. The coating can provide a moisture barrier around the chewable base core.

[0031] The present invention further provides a method to improve physical performance in a mammal engaged on physical effort, comprising the steps of: i) providing one or more units of a chewable composition comprising a gum base and an amount of reduced glutathione; and ii) masticating by the mammal the one or more units of the chewable composition for a time sufficient to release a quantity of the reduced glutathione into the oral cavity of the mammal, for absorption into the blood; thereby improving the physical performance in the mammal engaged on physical effort.

[0032] The present invention further provides a method to reduce blood lactate concentration in a mammal that has performed intense physical effort, comprising the steps of: i) providing one or more units of a chewable composition comprising a gum base and an amount of reduced glutathione; and ii) masticating by the mammal the one or more units of the chewable composition for a time sufficient to release a quantity of the reduced glutathione into the oral cavity of the mammal, for absorption into the blood; thereby reducing blood lactate concentration in the mammal that has performed intense physical effort.

[0033] In an aspect of the invention, the concentration of the reduced glutathione in the composition is from 1,000 to 150,000 ppm.

[0034] In another aspect of the invention, the acute GSH depletion is produced by alcohol ingestion, or by exercise, particularly intense exercise, in mammals and humans, particularly humans.

[0035] In an aspect of the invention, an acute depleting event can be a condition of the mammal or human, or of the environment, which can hasten the depletion of GSH in the body for up to 3 days after the onset of the acute depleting event. The acute depleting event hastens depletion of GSH beyond the normal daily depletion of GSH. The hastening of the depletion of GSH in the body can also continue for up to two days, including up to one day, up to 12 hours, up to three hours, up to two hours, up to one hour, up to 30 minutes, up to 10 minutes, and up to 5 minutes. [0036] An acute depleting event can also be a condition of the mammal or human, or of the environment, which begins hastening the depletion of GSH in the body within 1 hour, including within 30 minutes, and within 5 minutes, including an almost immediate hastening of GSH depletion, after the onset of the acute depleting event.

[0037] An acute depleting event can be the consumption of ethyl alcohol, intense exercise, stress, migraines, administration of a drug or medicament, including the administration of acetaminophen, an influence, an exposure to or consumption of a heavy metal, a bacterial or viral infection, an event that results in an accumulation of lactic acid in the blood, and an event that results in an increase in free radicals into the cells of the mammal.

[0038] In an aspect of the invention, the method herein, including the masticating of the one or more units of the chewable composition for absorption of GSH into the blood, begins reducing the hastened depletion of glutathione in the mammal within 20 minutes, including within 10 minutes, and including within 5 minutes after the onset of mastication, and including an almost immediate reduction in the hastened depletion of GSH after the onset of mastication.

[0039] In a further aspect, the masticating of the one or more units of the chewable composition is initiated within 3 days, including within 2 days, within 1 day, 12 hours, and within 6 hours, of an onset of the acute depleting event.

[0040] In another aspect of the invention, the masticating of the one or more units of the chewable composition is initiated within 3 hours, including within 2 hours, within 1 hour, 30 minutes, 15 minutes, and 5 minutes, of an onset of the acute depleting event.

[0041] In another aspect of the invention, the time for masticating is at least one minute, and preferably up to about 10 minutes.

[0042] In a further aspect of the invention, the resulting reduction in the acute depletion of glutathione in the mammal when masticating the quantity of the reduced glutathione, is a greater reduction than that when instead a same quantity of reduced glutathione is ingested orally.

[0043] In another aspect of the invention, the amount of the reduced glutathione in a dose of one or more of the units of the chewable composition is at least 30 mg, including between 50 and 800 mg, including at least 100 mg, preferably between about 100 and 600 mg, preferably between about 200 and 400 mg, and more preferably at least about 300 mg. [0044] In an aspect of the invention the time for masticating is at least one minute, and preferably up to about 10 minutes.

Detailed Description of the Invention:

[0045] The administration and mastication of the chewable compositions comprising glutathione described in the invention transport the glutathione through the oral mucosa and into the blood. Without being bound by any particular theory, it is believed that the glutathione absorbed into the blood immediately begins to be taken up into any and all cells of the body. Consequently, measuring the glutathione concentration in the plasma or serum is an inaccurate measure or indication of the level of glutathione in the body, or its level or rate of depletion or recovery. It is believed that at any time up to 95%, or more, of the glutathione in the body is contained within cells. While the body experiences a natural depletion of GSH in the course of normal daily living, acute depletion events as described herein can hastened or increase the depletion of GSH. While the body's native production of GSH attempts to keep up with and replace the depleting amounts and levels of GSH, the invention provides a safe, convenient, efficient and effective composition and method for supplementing the replenishment of GSH before or during a period of GSH depletion, including hastened depletion, by the administration of the GSH in a chewable base composition.

[0046] In an aspect of the present invention the chewable base comprises: a water- insoluble gum base selected from the group of suitable natural and synthetic elastomers and rubbers; a bulking water-soluble portion comprising about 10%-70% of sugars or polyalcohol in powder form, selected from the group consisting of, but not limited to, sucrose, dextrose, maltose, dextrin, dried invert sugar, fructose, levulose, galactose, corn syrup solids and the like, alone or in combination, and powder sugar alcohols selected from the group consisting of, but not limited to, sorbitol, erythritol, xylitol, mannitol, maltitol, isomalt and combinations thereof; 0.1 - 6.0% of softeners and plasticizers selected from the group consisting of, but not limited to, glycerin, oils, waxes of fats, including but not limited to hydrogenated vegetable oils soybean oil, cottonseed oil, palm oil, palm kernel oil, coconut oil, sunflower oil, corn oil, captrin, liquid lipids made from triglycerides of fatty acids and mixtures thereof, partially hydrogenated vegetable oils, solid lipids made from triglycerides of fatty acids and mixtures thereof, beeswax, microcrystalline wax, carnauba wax, in combination. The chewable base also can comprise one or more emulsifier comprising about 0,01- 5% by weight, more preferably 0,5 - 2% by weight, thereof, selected from the group consisting of, but not limited to, lecithin, diglycerides of fatty acids, glycerol mono and distearate, glycerol triacetate, acetylated monoglyceride, sugar and polyol esters, and mixtures thereof, one or more high impact sweeteners selected from the group consisting of, but not limited to, acesulfame, aspartame, sucrose, stevia, fructose, maltose, glucose, saccharine and combinations thereof, and the use of one or more suitable flavoring agents selected from the group consisting of natural or artificial sources in liquid or powder form.

Models for assessing; GSH supplementation efficacy during; acute GSH depletion:

Ethyl alcohol detoxification

[0047] In average, the human body is able to metabolize ethyl alcohol at a rate of 7-9 grams per hour. (Alcohol metabolism. Arthur I Cederbaum, PhD. Clin Liver Dis. 2012 Nov; 16(4): 667-685.) As GSH appears to be a key molecule for the metabolization of ethanol, an effective GSH supplementation might have a traceable impact on the rate at which ethanol is metabolized.

[0048] The administration of the chewable compositions comprising GSH described in the invention can increase the alcohol metabolization rate by more than 50% in healthy individuals, within a period of time of one hour or less, including 30 minutes, 15 minutes, 10 minutes and 5 minutes, using a low dose of at least about 30 mg, and including about 100 mg and about 300 mg of GSH.

Lactic acid metabolization and fatigue perception associated to intense exercise

[0049] As GSH appears to be a key molecule during intense exercise, an effective GSH supplementation might have a traceable impact on the fatigue perception and blood lactate concentration, during and after intense exercise. The chewable compositions described in the invention, using a low dose of 200 to 300 mg, have been shown to significantly decrease the fatigue perception in healthy individuals during or after intense exercise. Moreover, this effect was exerted in a very short time. [0050] The chewable composition No.1 described herein using a low dose of GSH (240 mg) improve significantly physical performance in healthy individuals engaged in intense physical effort by extending the limit time to exhaustion (Tlim) during a supramaximal exercise test, and significantly reduce the blood lactate concentration associated to intense physical effort during the same.

[0051] As shown, the compositions were able surprisingly to produce a traceable impact on both the alcohol-related and exercise-related depletion models. Therefore, the chewable compositions described in this invention are an effective delivery system for GSH and can be used by healthy individuals during circumstances of acute GSH depletion.

[0052] At any time in the body, up to 95% of the GSH in the body is contained within the cells of the body, include red blood cells and white blood cells. While the plasma and serum may contain GSH being transported within the blood, such levels are not a reliable or predictable measure of GSH level, or of the level of depletion. While methods exist for determining the level of GSH in cells or whole blood, in the references described and incorporated herein, good evidence of the effects of GSH depletion, and of the recovery of GSH levels, can be determined by the improvement in the effects or the performance of the human condition.

Examples

Example 1 : Preferable compositions

[0053] The following compositions were prepared in order to assess the efficacy of the administration of a chewable composition comprising GSH in healthy individuals during acute GSH depletion situations.

[0054] An amount of gum base (provided by Gum Base Co.) of the formula was melted in a chewing gum double-sigma blade kneader thermostated at 50° C. Once the gum base is melted, 50% of sorbitol powder and 50%> of mannitol powder (both products provided by Roquette Corporate) were added to the kneader and mixed for 10 minutes with the gum base. Once the previous mixture was homogeneous, 50% of the glycerol and liquid sorbitol were added and after a further 10 minutes, the 50% of the sorbitol powder and the total Sucralose was added and mixed for 10 minutes. The softener, the rest of the glycerol and liquid sorbitol were added and mixed by 10 minutes. After the previous mix was homogeneously mixed, the temperature was reduced to 35°C by using a cooling jacket. Once the temperature was set to 35°C the flavor, the softener and menthol were added and mixed for 5 minutes. Finally, the L-Glutathione (Kohjin Life Sciences Co. Ltd) was added and mixed for 10 additional minutes until homogeneous.

Table 1. Chewable Base Core Compositions, per piece

Placebo Composition No.l Composition No.2

Ingredient mg Percentage mg Percentage mg Percentage

Gum base 500 33.3 500 33.3 500 33.3

Sorbitol Powder 268.4 17.9 238.4 15.9 218.4 14.6

Sorbitol liquid 55 3.7 55 3.7 55 3.7

Glycerin 40 2.7 40 2.7 40 2.7

Mannitol 60 4.0 60 4.0 60 4.0

Mint Flavor

20 1.3 20 1.3 20 1.3

liquid

Menthol 10 0.7 10 0.7 10 0.7

Softener 10 0.7 10 0.7 10 0.7

Sucralose 6.6 0.4 6.6 0.4 6.6 0.4

L-Glutathione 0 0.0 30 2.0 50 3.3

Maltitol 530 35.3 530 35.3 530 35.3

Total 1500 100 1500 100 1500 100

[0055] The ingredients of the core composition were mixed, formed into core pieces, cooled at 20-25°C, and transported to the lamination equipment in which square core pieces of 1.1 g each were prepared. The subsequent pieces were coated using maltitol syrup in a similar process described in United States Patent Application 20060034975, the disclosure of which is incorporated by reference, in which maltitol syrup was crystallized in order to produce multiple layers of coating until gums reach 1.5g weight.

Example 2: GSH release test

[0056] In order to evaluate the efficient delivery of the GSH present on the chewable compositions during mastication of the same, a GSH release test was performed. For this purpose two pieces of composition No.l and two pieces of composition No.2 were chewed for a total of 10 minutes in separate tests. The total amount of saliva produced every 1 minute of mastication was collected in separate tubes, and the content of GSH present on each sample was subsequently analyzed by colorimetric method in spectrophotometer UV mini Shimadzu 1240 at 405nm, using Ellman's reagent 5,5'- Dithio-bis-(2-nitrobenzoic acid). Calibration curve was performed using L-glutathione 98% w/w provided by Sigma Aldrich as standard. The percentage of GSH released from the chewable composition during each minute of mastication is show in Table 2.

[0057] Table 2 shows the percentage of GSH released from composition No.l and composition No.2 at every minute during the 10 minute period of mastication. Tests were performed in triplicate.

Table 2.

GSH release during mastication

Composition No.1 Composition No.2

Minutes

(Release %) (Release %)

1 20 15

2 35 36

3 65 62

4 85 82

5 95 93

6 96 94

7 97 96

8 97 96.5

9 97.5 97

10 98 97.5

Example 3: Ethyl alcohol detoxification as a model for assessing GSH chewable composition supplementation

[0058] Fifteen (15) healthy adults (32 years old of average age, and occasional alcohol drinkers) participated in four trials of ethanol detoxification performed in four non- consecutive days. Individuals followed a control diet and daily routine for each day during the protocol. Each subject had a minimum period of 5 days without alcohol consumption before the trial.

[0059] Each trial was performed administering 100 ml of whisky (40% v/v; approximately 32 grams of pure ethanol) in a period of 10 minutes. Immediately after alcohol ingestion, blood alcohol concentration was measured every 15 minutes using a breathalyzer (Alcoscan AL7000) during the following 4 hours after alcohol ingestion.

[0060] In order to compare the values of blood alcohol concentration estimated by the breathalyzer device and the effective blood alcohol concentration, blood samples were collected in two subjects at 40, 80, 120 and 160 minutes after alcohol ingestion on days 1 and 3, samples were subsequently analyzed in order to determine the alcohol concentration in blood.

[0061] During each trial day individuals were administered with the compositions as follow:

Trial day 1 : placebo (6 pieces)

Trial day 2: Composition No.l (6 pieces)

Trial day 3: composition No.2 (6 pieces)

Trial day 4: 50 ml of GSH solution 0.6% w/v.

[0062] Chewable compositions (placebo and compositions 1 and 2) were administered in a series of doses, one piece per dose. Chewing time for each dose was 10 minutes, starting 10 minutes before alcohol ingestion and continuing each 10 minutes, finishing 50 minutes after. In the trial leg of GSH solution ingestion, the GSH solution was ingested immediately after alcohol.

[0063] Results of Table 3 show the average BAC concentration measured by breathalyzer Alcoscan A17000 during a period of four hours in 15 subjects after administration of 100ml of whisky (40% v/v; approximately 32 grams of pure ethanol). Significant improvements in the rate of alcohol metabolization were observed with composition No.l and composition No.2. The results for alcohol metabolization rate are shown in Table 4 (detoxification is consider finished when a level of 0.05 g/L or less is achieved using the breathalyzer Alcoscan A17000).

[0064] Table 5 shows the average blood alcohol concentration measured in blood and breathalyzer for two subjects in trial day 1 and trial day 3. Significant improvements in the alcohol metabolization rate were observed on trial day 3 (Composition No.2). The results for alcohol metabolization rate are shown in table 6 (detoxification is consider finished when a level of 0.05 g/L or less is achieved)

Table 3.

Comparison of four compositions on Average BAC measured by Breathalyzer (g/L) in 15 subjects

Time (min) Placebo GSH Solution Composition 1 Composition 2

0.6% w/v

0 0.00 0.00 0.00 0.00

20 0.21 0.23 0.20 0.19

30 0.30 0.31 0.26 0.24

45 0.34 0.30 0.25 0.25

60 0.30 0.27 0.23 0.21

75 0.27 0.25 0.18 0.16

90 0.26 0.23 0.15 0.12

105 0.24 0.22 0.12 0.10

120 0.23 0.20 0.10 0.07

135 0.20 0.16 0.08 0.03

150 0.19 0.13 0.04 0.02

165 0.11 0.10 0.00 0.00

180 0.10 0.08 0.00 0.00

195 0.10 0.05 0.00 0.00

210 0.04 0.00 0.00 0.00

225 0.01 0.00 0.00 0.00

240 0.00 0.00 0.00 0.00

Table 4.

Alcohol metabolization rate (g/h) in 15 subjects

using breathalyzer

Placebo 9 GSH solution 0.6% w/v Composition 1 Composition 2

Table 5.

Average BAC in blood and breathalyzer (g/L) for 2 subjects

Time Placebo Placebo Comp. 2 Comp. 2 (minutes) Breathalyzer Blood Breathalyzer Blood

0 0 0 0 0

40 0.36 0.37 0.3 0.31

80 0.27 0.253 0.2 0.22

120 0.2 0.22 0.1 0.12

160 0.1 0.11 0.05 0.06

Table 6.

Alcohol metabolization rate (g/h) in 2 subjects in blood

and breath

Composition Breathalyzer Blood

Placebo 9.0 8.9

Composition 2 12 12

Example 4: Intense exercise-related acute GSH depletion as a model for assessing GSH chewable composition supplementation

[0065] Thirteen healthy adults participated in four different trials in order to evaluate the effectiveness of chewable GSH composition in healthy individuals during acute intense exercise related GSH depletion. Subjects followed a control diet and daily routine in the days that the exercise test was performed.

[0066] Individuals were randomly divided into four groups as detailed below in order to perform the following exercise trials:

Group 1 : Maximal effort test

[0067] Four healthy subjects performed an incremental treadmill exercise test in two non- consecutive days.

[0068] Each trial day subject ran on a treadmill set at a fixed slope of 0 at an initial speed of 9 km/h, followed by an increase of 1 km/h and 1° slope every two minutes until subjects could not maintain the work required (having reached their maximal exercise capacity before fatigue). Subjects had a 30 minutes recovery rest. During this period individuals were administered with each composition as follow: Day 1, placebo (6 pieces); Day 2, Composition No.2 (6 pieces).

[0069] After the recovering time (30 minutes), a second round of incremental treadmill exercise was performed using the same protocol.

[0070] Immediately after exercise, subjects were asked to complete a fatigue self-perception questionnaire in which individuals rated their fatigue perception in a scale from 1 to 4 in which 4 is the maximum for fatigue perception and 1 is the minimum (results are shown in Table 7).

Table 7.

Self-rated fatigue perception after maximal effort test in 4

subjects

Placebo Composition No.2

Subject 1

Subject 2

Subject 3

Subject 4

Group 2: 100m swimming test

[0071] Two subjects performed an exercise test consisting on 100 meters of swimming at their maximal speed in 2 non-consecutive days. Before each trial subjects were administered with each composition as follow: Day 1, placebo (4 pieces); Day 2, Composition No.2 (4 pieces). Compositions were chewed for a period of 20 minutes (2x2 pieces, 10 minutes each) immediately before each exercise test.

[0072] Immediately after exercise subjects were asked to complete a fatigue self-perception questionnaire in which individuals rated their fatigue perception in a scale from 1 to 4, in which 4 is the maximum for fatigue perception and 1 is the minimum (results are shown in Table 8).

Table 8.

Average self-rated fatigue perception after swimming

test in 2 subjects

Placebo Composition No.2

Subject 5 4 2

Subject 6 4 3

Group 3 : HUT recovery test

[0073] Four subjects performed, in three non-consecutive days separated one week from each other, a high intensity interval training (HUT) recovery trial. The trial consisted of 10 x 1 min intervals at 85-95 % aerobic power (each interval followed by 30 seconds at 20-30% aerobic power). After each trial, subjects were administered with each composition as follow: Day 1, placebo (6 pieces); Day 2, Composition No.2 (6 pieces); Day 3, 50ml GSH solution 0.6% w/v.

[0074] Compositions were consumed in a period of 30 minutes after the trial divided in doses of two pieces (each dose chewed for 10 minutes).

[0075] Immediately after the trial, subjects were asked to complete a fatigue self-perception questionnaire in which individuals rated their fatigue perception/recovery in a scale from 1 to 4, in which 4 is the maximum for fatigue perception and 1 is the minimum (results are shown in Table 9).

[0076] The results indicate that the chewable Composition No.2 was able to inhibit fatigue perception and improve recovery after high intensity interval training.

Table 9.

Self-rated fatigue perception after HUT test in 4 subjects Placebo Composition

No.2

Subject 7 4 2

Subject 8 4 3

Subject 9 4 2

Subject 10 4 3

Group 4: Double supramaximal running test

[0077] Three healthy subjects (mean age = 30) performed an exercise trial in order to evaluate the effect of composition No.l on blood lactate concentration and physical performance related to intense exercise.

[0078] During a period of 4 weeks, subjects performed one supramaximal exercise trial per week. After the trial, subjects return to their normal activities with no exercise during the following 6 days.

[0079] The supramaximal exercise consisted in 4 consecutive intervals as follows:

[0080] Warming: The first interval consisted in a warming running on a treadmill

(Technogym Excite) for 6 minutes at a speed of 7.5 km/h.

[0081] First exhaustion test: Immediately after warming, subj ects completed a supramaximal continuous running time limit (Tlim) exercise test performed at 130% Vomax until exhaustion (second interval).

[0082] Active resting: Immediately after, an active resting interval (third interval) was performed consisting on a treadmill walk at 4.5 km/h for 16 minutes. During this interval a total of eight pieces of placebo or composition No.l (240 mg of GSH) were administered in consecutive doses of two pieces chewed each for 4 minutes.

[0083] Second exhaustion test: Immediately after the active resting, subjects repeated another supramaximal continuous running time limit (Tlim) exercise test performed at 130% Vomax until exhaustion (fourth interval).

[0084] Composition No.l and placebo were administered as follows:

Week 1: 8 pieces of composition No.l administered during active resting (third interval).

Week 2: 8 pieces of placebo administered during active resting (third interval) Week 3: 2 pieces of composition No.l administered every day for seven days plus 8 pieces of composition No.l administered during active resting (third interval).

Week 4: 2 pieces of composition No.l administered every day for seven days plus 8 pieces of composition No.l administered during active resting (third interval).

[0085] Blood lactate was measured using Lactate Plus (Nova Medical) 2 minutes after each exhaustion test and 11 minutes after the second exhaustion test. Heart rate, autonomic activity and heart rate variability was monitored using a Polar Wear Link band. Data collected was processed by Kubios HRV 2.1 software provided by the Biocenter of the University of Kuopio in Finland.

[0086] The results shown in Table 10 demonstrate that Composition No.l was able to significantly (almost 50% in week 4 compared to placebo) extend the exercise time to exhaustion (Tlim).

[0087] The results shown in Table 11 demonstrate that composition No.l was able to significantly reduce blood lactate concentration associated to intense physical effort. Week 4 showed a blood lactate average reduction of 37% during Tlim 2 and 22% in post (11 minutes) intense physical effort compared with placebo.

Table 10

Limit time at 130% Vomax (min : seg)

Week 1 Week 2 Week 3 Week 4

Tlim l Tlim 2 Tlim 1 Tlim 2 Tlim l Tlim 2 Tlim 1 Tlim 2

Subject 1 6:10 4:10 6:10 3:19 6:07 4: 17 6:06 4:24

Subject 2 3:00 2:30 3:01 1 :53 3:00 2:10 3: 10 2:50

Subject 3 5:10 4:00 5:11 3:00 5:10 4:40 5:50 5:00

Table 11

Blood lactate (mmol IV)

Week l Week 2 Week 3 Week 4

Tlim Tlim Tlim Tlim Tlim Tlim Tlim Tlim

Post Post Post Post 1 2 1 2 1 2 1 2 Subject 1 16.9 21.9 12.0 16.2 22.4 12.0 15.7 17.9 11.5 16.0 17.5 11.2 Subject 2 14.0 17.0 8.0 14.7 19.5 10.0 14.0 16.0 8.0 13.4 14.0 7.7 Subject 3 13.0 18.0 10.0 14.2 21.0 12.0 12.1 16.1 10.0 12.4 14.5 9.0

[0088] All journal publications and patent and patent application publications listed herein are incorporated by reference in their entireties.