WO2023016852A1 - Amino acid composition as a vasodilating agent - Google Patents

Amino acid composition as a vasodilating agent Download PDF

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Publication number
WO2023016852A1
WO2023016852A1 PCT/EP2022/071622 EP2022071622W WO2023016852A1 WO 2023016852 A1 WO2023016852 A1 WO 2023016852A1 EP 2022071622 W EP2022071622 W EP 2022071622W WO 2023016852 A1 WO2023016852 A1 WO 2023016852A1
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salt
arginine
citrulline
glutathione
norvaline
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PCT/EP2022/071622
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French (fr)
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Andrea Salmaso
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Andrea Salmaso
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/08—Vasodilators for multiple indications
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
    • A23L2/385—Concentrates of non-alcoholic beverages
    • A23L2/39—Dry compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/06—Tripeptides
    • A61K38/063—Glutathione
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0087—Galenical forms not covered by A61K9/02 - A61K9/7023
    • A61K9/0095—Drinks; Beverages; Syrups; Compositions for reconstitution thereof, e.g. powders or tablets to be dispersed in a glass of water; Veterinary drenches

Definitions

  • the present invention relates to a composition for oral subministration, as food supplementation or medical purposes and, in particular, to a composition comprising amino acids, which is effective as a vasodilator and/or in a method for increasing blood flow in skeletal muscle.
  • Nitric oxide (or nitric monoxide, chemical formula: NO) is a gas playing a crucial role in the human health, particularly by acting as a mediator and activator of blood vessel dilation, a physiological process that promotes the supply of blood, oxygen and nutrients to tissues, as well as the removal of metabolic wastes from tissues.
  • nitric oxide is well known among sportsmen.
  • its properties of increasing blood supply in skeletal muscle and, thus, enabling better athletic performance are well recognize among scientists as well as in the marketplace and sportsmen taking food supplements for ergogenic purposes.
  • Skeletal muscle is the biggest organ in the entire body. It enables the movement of the human body and has a large energy and oxygen consumption, especially under physical exertion. Therefore, it is extensively supplied with blood vessels in order to meet its energy needs.
  • the augmentation of blood supply to the muscle means increased supply of nutrients and oxygen that can be used for energy, as well as an increased capacity to dispose of metabolic waste produced during physical activity.
  • the first food supplements for the stimulation of nitric oxide production were based on L-Arginine.
  • L-Arginine is a proteinogenic amino acid (i.e. it is used in the human body as a monomer for protein synthesis). It is defined as a semi-essential (or conditionally essential) amino acid in humans, as under certain conditions (stress, certain diseases, intense and frequent physical activity) the body's endogenous production may not meet requirements. L-Arginine is also a metabolically versatile amino acid as it acts as a precursor for various metabolic intermediates (e.g. creatine, urea) and plays a key role in nitric oxide synthesis as it acts as a precursor for nitric oxide itself.
  • various metabolic intermediates e.g. creatine, urea
  • Nitric oxide is synthesized in the endothelial cells of blood vessels by the enzyme (i.e. a biological catalyst) Nitric Oxide Synthase (NOS).
  • NOS Nitric Oxide Synthase
  • L-Arginine into the nitric oxide and L-Citrulline.
  • L-Citrulline via the nitric oxide cycle, is converted back to L- Arginine; on the other hand, nitric oxide produced diffuses across the endothelial cell membrane and reaches the neighboring smooth muscle cells of blood vessel: here the nitric oxide initiates a series of reactions that culminate in smooth muscle relaxation. This relaxation of the muscle cells results in vasodilation of the blood vessel and, therefore, increased blood supply by the vessel itself.
  • L-Arginine is 40% destroyed by the small intestine and in the “first-pass metabolism” into the liver. This is due to the activity of an enzyme called Arginase, present in both the small intestine and liver, which converts L-Arginine into ornithine and urea (Guoyao Wu, Amino Acids 2009 May;37(1):1 - [0010]
  • Arginase is also present in these cells and again 'sequesters' the L-Arginine into the so-called urea cycle, in which L-Arginine is converted to ornithine and urea.
  • L-Citrulline Originally identified in 1914 from the watermelon (Citrullus lanatus), from which it takes its name, L-citrulline is a non-essential, non-proteinogenic amino acid naturally present in the human body.
  • L-Citrulline is a precursor to L-Arginine and, differently to the latter, it is not subject to degradation by the enzyme Arginase, either in the intestine or in the endothelial cells of blood vessels. L-Citrulline therefore has a higher bioavailability than L-Arginine, as the body absorbs it more efficiently once taken orally. As it reaches the endothelial cells of blood vessels it can be converted to L-Arginine, thus participating in the synthesis of nitric oxide.
  • L-Citrulline alone cannot completely replace the functions of L-Arginine. Indeed, L-Citrulline has been shown to be more effective when administered together with L-Arginine. Noteworthy, the combination of the two amino acids in a 1 :1 ratio is more effective in stimulating nitric oxide synthesis than using each amino acid alone (Masahiko Morita, Toshio Hayashi, Masayuki Ochiai, Morihiko Maeda, Tomoe Yamaguchi, Koichiro Ina, Masafumi Kuzuya, Biochem Biophys Res Commun 2014 Nov 7;454(1 ):53-7). This is due to L-Citrulline ability to allosterically inhibit the enzyme Arginase both in the small intestine and in liver.
  • L-Arginine and/or L-Citrulline are present in compositions having a vasodilator effect even in the presence of other formulations, such as in patent application n. RU2464019C1 , disclosing a composition having an endothelium-protective, vasodilator, and angio-protective effect, containing a NO donors such as L-arginine or L-citrulline and an Arginase inhibitor selected from L-Norvaline and nor-NONA.
  • L-Norvaline (a-aminovaleric acid) is a non-essential non-proteogenic amino acid which is an inhibitor of the enzyme Arginase in the human body (R. Rognstad, Biochim Biophys Acta 1977 Feb 28;496(2):249-54). It is slows down the ability of Arginase to capture Arginine within the urea cycle. L-Norvaline also inhibits the enzyme Ornithine
  • Transcarbamylase further inhibiting the urea cycle (Hans-Peter Bode, Bernadette Eder, Michael Trautmann, European Journal of Biochemistry, June 1994222(3):69-877).
  • Glutathione is a tripeptide composed of the amino acids L-glutamate, L-cysteine, and glycine. Glutathione is an extremely potent antioxidant and detoxifying agent ubiquitously present in cells’ cytoplasm in the human body. It is able to inactivate the free radicals produced in the cells and to inhibit their ability to react and damage membranes and other biomolecules present in the cell, such as proteins and nucleic acids.
  • Glutathione is effective in increasing the body's stores of Glutathione (John P Richie Jr, Sailendra Nichenametla, Wanda Nschreib, Ana Calcagnotto, Jeremy S Haley, Todd D Schell, Joshua E Muscat, Eur J Nutr 2015 Mar;54(2):251 -63).
  • Glutathione has the ability to upregulate the action of nitric oxide. Indeed, nitric oxide has a relatively short half-life and is subject to degradation by reaction with free radicals. Glutathione is able to deactivate free radicals, thus shielding and protecting nitric oxide from oxidative damage and enhancing the shelf-life and, thus, the effectiveness of nitric oxide (O Pechanova 1 , M Kashiba, M Inoue, Jpn J Pharmacol 1999 Oct;81 (2):223-9). In addition, upon interaction with nitric oxide, Glutathione forms S-nitrosoglutathione, stabilizing nitric oxide itself and further protecting it from free radicals attack. S-nitrosoglutathione is then able to release and liberate nitric oxide via reconversion to free glutathione and nitric oxide (Shigeru Oae & Kbichi Shinhama, The
  • a composition containing L-Citru 11 i ne or a salt thereof and Glutathione or a salt thereof is the subject of patent application WO2018221654A1 for use in increasing the ratio of muscle mass to total body weight in combination with a resistance workout program.
  • Sildenafil citrate as well as all artificial substances with pharmacological activity, possesses side effects: dizziness, flushing, gastroesophageal reflux, headache, and dyspepsia among the most common (K E Andersson, Br J Pharmacol 2018 Jul;175(13):2554-2565).
  • the issue addressed by the present invention is to provide a new composition useful for stimulating nitric oxide production with molecules already present in nature and free of adverse side effects.
  • the invention relates to a composition to be used as a vasodilator and/or in a method for increasing blood flow in skeletal muscle.
  • Figure 1 shows the kinetics of nitric oxide production in HMVEC cells (Human MicroVascular Endothelial Cells).
  • Figure 2 shows nitric oxide production calculated after the addition of L-Norvaline or Glutathione (where applicable) (at minute 60' of the experiment), i.e. from the time when L-Arginine L-Citrulline, L-Norvaline and Glutathione are simultaneously present in the “Norv GSH” condition.
  • Figure 3 illustrates the VAS (visual analogue scale) scores of the five questions of the questionnaire evaluating the combined effect of L-Arginine or a salt thereof, L-Citrulline or a salt thereof, Glutathione and L-Norvaline or a salt thereof for each group of amateur athletes following training after taking placebo or after taking one of the four treatment mixtures:
  • GSH oral supplementation with mixture consisting of Glutathione
  • Figure 4 shows the change in VAS scores in each group of amateur sportsmen and sportswomen to the questions in the questionnaire assessing the combined effect of L- Arginine or a salt thereof, L-Citrulline or a salt thereof, Glutathione and L-Norvaline or a salt thereof after placebo intake and after intake of one of the four treatment mixtures: Arg + Cit mixture, Norv mixture, GSH mixture, Arg + Cit + Norv + GSH mixture.
  • An object of the present invention is a composition comprising L-Arginine or a salt thereof, L-Citrulline or a salt thereof, L-Norvaline or a salt thereof, and Glutathione.
  • composition comprising L-Arginine or a salt thereof, L-Citrulline or a salt thereof, L-Norvaline or a salt thereof, and Glutathione can indeed be effectively employed in medicine, as a medicament.
  • This composition has in fact been found to be particularly effective in the treatment of the human or animal body as a vasodilator and/or in a method of increasing blood flow in skeletal muscle.
  • composition comprising L-Arginine or a salt thereof, L-Citrulline or a salt thereof, L-Norvaline or a salt thereof and Glutathione is effective as a vasodilator and, therefore, can be effectively employed as a vasodilating agent and/or in a method to increase blood flow in skeletal muscle.
  • composition comprising L-Arginine or a salt thereof, L-Citrulline or a salt thereof, L-Norvaline or a salt thereof, and Glutathione for use as a vasodilator and/or in a method for increasing blood flow in skeletal muscle is preferred.
  • the salt of L-Arginine is chosen from: L- Arginine Hydrochloride, L-Arginine a-Ketoglutarate, L-Arginine Pyroglutamate, L- Arginine L-Aspartate, Di-L-Arginine Malate.
  • the salt of L-Citrulline is L-Citrulline Malate.
  • the amount of L-Arginine or a salt thereof is from 0.1 to 5 grams
  • the amount of L-Citrulline or a salt thereof is from 0.1 to 5 grams
  • the amount of Glutathione is from 0.01 to 0.50 grams
  • the amount of L-Norvaline or a salt thereof is from 0.1 to 5 grams, per dose.
  • the percentage of L-Arginine or a salt thereof is from 0.65 to 45.0 % (w/w)
  • the percentage of L-Citrulline or a salt thereof is from 0.65 to 50.0 % (w/w)
  • the percentage of Glutathione is from 0.065 to 20.0 % (w/w)
  • the percentage of L-Norvaline or a salt thereof is from 0.65 to 35.0 % (w/w).
  • It is another object of the invention to provide a food supplement comprising a composition including L-Arginine or a salt thereof, L-Citrulline or a salt thereof, L- Norvaline or a salt thereof, and Glutathione, including the preferred forms of implementation described above, in powder or tablet or capsule or soft capsule or liquid or gel or bar form.
  • It is another object of the invention to provide a pharmaceutical composition comprising L-Arginine or a salt thereof, L-Citrulline or a salt thereof, L-Norvaline or a salt thereof, and Glutathione, including the preferred forms of realization described above, and at least a pharmaceutically acceptable excipient.
  • Such pharmaceutical composition may be in solid form, i.e. in tablet or capsule form, or in liquid form, i.e. in the form of an injectable solution or syrup.
  • the pharmaceutical composition comprising L-Arginine or a salt thereof, L-Citrulline or a salt thereof, L-Norvaline or a salt thereof, and Glutathione, including the preferred realizations described above, and at least a pharmaceutically acceptable excipient is to increase blood flow in skeletal muscle.
  • Another object is the composition described above, including the various embodiments, for use in medicine.
  • another object is the aforementioned composition for use in a method of treating the human or animal body as a vasodilator and / or in a method for increasing blood circulation in the skeletal muscle.
  • composition described above is a food supplement.
  • composition according to the present invention (Leibovitz Norv GSH group) as a vasodilating agent and/or in a method for increasing blood flow in skeletal muscle.
  • Table (I) indicates that there is no difference between CN group (negative control; observed combination: L-Arginine + L-Citrulline) and Norv group (observed combination: L-Arginine + L-Citrulline + L-Norvaline), while the fluorescence of the nitric oxide probe in the simultaneous presence of L-Arginine, L-Citrulline, glutathione and L-Norvaline (condition 'Leibovitz Norv GSH') is statistically significant (p ⁇ 0.0001 ) compared to all other three conditions.
  • Example 1 Evaluation of the combined effect of L-Arginine or a salt thereof, L-Citrulline or a salt thereof, Glutathione and L-Norvaline or a salt thereof in immortalized Human MicroVascular Endothelial Cells (HMVEC) cultures.
  • HMVEC Human MicroVascular Endothelial Cells
  • HMVEC cells were chosen because they are one of the main sites of nitric oxide production in the body.
  • HMVEC cells were grown in M199 culture medium supplemented with FBS and growth factors and loaded into Leibovitz L-15 culture medium with DAF-FM DA probe (abeam: ab145295) 15 pM for 15'.
  • the DAF-FM DA probe is internalized by the cells and, once inside, is able to react with nitric oxide to produce a fluorescent compound. Fluorescence emission is therefore proportional to the nitric oxide produced in the cell.
  • CN negative control
  • L-Arginine 2mM + L-Citrulline 2mM for 1 h.
  • Norv HMVEC cells maintained in Leibovitz culture medium with L-Arginine 2mM + L- Citrulline 2mM for 1 h. After 30', L-Norvaline 2mM was added for a further 30'. Combination observed: L-Arginine + L-Citrulline + L-Norvaline.
  • GSH HMVEC cells maintained in Leibovitz culture medium with L-Arginine 2mM + L- Citrulline 2mM for 1 h. After 30', Glutathione 2mM was added for a further 30'. Combination observed: L-Arginine + L-Citrulline + Glutathione.
  • Norv GSH HMVEC cells maintained in Leibovitz culture medium with L-Arginine 2mM + L-Citrulline 2mM for 1 h. After 30', L-Norvaline 2mM + Glutathione 2mM was added for a further 30'. Combination observed: L-Arginine + L-Citrulline + L-Norvaline + Glutathione.
  • Example 2 Evaluation of the combined effect of L-Arginine or a salt thereof, L-Citrulline or a salt thereof, Glutathione and L-Norvaline or a salt thereof in amateur athletes.
  • Participants recruitment subjects participating in the trial evaluating the efficacy of the invention were selected as follows: healthy subjects, of both sexes, aged between 20 and 55 years, who habitually practiced (at least twice a week) amateur or professional sports activities lasting between 1 h and 2h, and falling within the following disciplines: body building, gym fitness classes, crossFit®, calisthenics, martial arts and combat sports, team sports (football, futsal, volleyball, rugby, basketball).
  • each participating subject was required to take a powder mixture dissolved in water (100- 250 mL) 45'-60' before training for two workouts. Following each workout, each participant was asked to complete an evaluation questionnaire.
  • Each participant received, in one training session, a 'treatment' mixture containing one or more of the substances L-Arginine or a salt thereof, L-Citrulline or a salt thereof, L- Norvaline or a salt thereof and Glutathione and, in another training session, a 'placebo' mixture containing none of the above substances but with an appearance, smell and taste similar on the ‘treatment’ mixture.
  • GSH containing Glutathione
  • Arg + Cit + Norv + GSH containing L-Arginine L-Aspartate, L-Citrulline, L-Norvaline and Glutathione.
  • Evaluation questionnaire The evaluation questionnaire completed by the study participants after training is as follows.
  • VAS Visual Analogue Scale

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Abstract

The present invention relates to a composition for oral subministration, as food supplementation or medical purposes and, in particular, to a composition comprising amino acids, which is effective as a vasodilator and/or in a method for increasing blood flow in skeletal muscle.

Description

AMINO ACID COMPOSITION AS A VASODILATING AGENT
Description
Field of Technology
[0001] The present invention relates to a composition for oral subministration, as food supplementation or medical purposes and, in particular, to a composition comprising amino acids, which is effective as a vasodilator and/or in a method for increasing blood flow in skeletal muscle.
State of the Art
[0002] Nitric oxide (or nitric monoxide, chemical formula: NO) is a gas playing a crucial role in the human health, particularly by acting as a mediator and activator of blood vessel dilation, a physiological process that promotes the supply of blood, oxygen and nutrients to tissues, as well as the removal of metabolic wastes from tissues.
[0003] In food supplementation field, nitric oxide is well known among sportsmen. In particular, its properties of increasing blood supply in skeletal muscle and, thus, enabling better athletic performance, are well recognize among scientists as well as in the marketplace and sportsmen taking food supplements for ergogenic purposes.
[0004] Skeletal muscle is the biggest organ in the entire body. It enables the movement of the human body and has a large energy and oxygen consumption, especially under physical exertion. Therefore, it is extensively supplied with blood vessels in order to meet its energy needs. The augmentation of blood supply to the muscle means increased supply of nutrients and oxygen that can be used for energy, as well as an increased capacity to dispose of metabolic waste produced during physical activity. [0005] In the past, the first food supplements for the stimulation of nitric oxide production were based on L-Arginine.
[0006] L-Arginine is a proteinogenic amino acid (i.e. it is used in the human body as a monomer for protein synthesis). It is defined as a semi-essential (or conditionally essential) amino acid in humans, as under certain conditions (stress, certain diseases, intense and frequent physical activity) the body's endogenous production may not meet requirements. L-Arginine is also a metabolically versatile amino acid as it acts as a precursor for various metabolic intermediates (e.g. creatine, urea) and plays a key role in nitric oxide synthesis as it acts as a precursor for nitric oxide itself.
[0007] Nitric oxide is synthesized in the endothelial cells of blood vessels by the enzyme (i.e. a biological catalyst) Nitric Oxide Synthase (NOS). NOS converts L-Arginine into the nitric oxide and L-Citrulline. L-Citrulline, via the nitric oxide cycle, is converted back to L- Arginine; on the other hand, nitric oxide produced diffuses across the endothelial cell membrane and reaches the neighboring smooth muscle cells of blood vessel: here the nitric oxide initiates a series of reactions that culminate in smooth muscle relaxation. This relaxation of the muscle cells results in vasodilation of the blood vessel and, therefore, increased blood supply by the vessel itself.
[0008] However, taking a dietary supplement containing L-Arginine alone in order to boost the nitric oxide endogenous production has strong limitations that make it scarcely effective.
[0009] Firstly, it should be considered that once taken orally, L-Arginine is 40% destroyed by the small intestine and in the “first-pass metabolism” into the liver. This is due to the activity of an enzyme called Arginase, present in both the small intestine and liver, which converts L-Arginine into ornithine and urea (Guoyao Wu, Amino Acids 2009 May;37(1):1 - [0010] In addition, the remaining portion of L-Arginine reaching the blood vessel endothelial cells is not entirely available for nitric oxide synthesis. Indeed, Arginase is also present in these cells and again 'sequesters' the L-Arginine into the so-called urea cycle, in which L-Arginine is converted to ornithine and urea.
[0011] In endothelial cells, therefore, there is competition between NOS and Arginase enzymes for the utilization of L-Arginine: the former enzyme converts L-Arginine to L-Citru 11 i ne and nitric oxide, the latter enzyme converts L-Arginine to ornithine and urea. Unfortunately, the conversion efficiency of Arginase is much higher than the conversion efficiency of NOS, as it has been calculated that Arginase is about a thousand times more active than NOS (Mihail V Pokrovskiy, Mihail V Korokin, Svetlana A Tsepeleva, Tatyana G Pokrovskaya, Vladimir V Gureev, Elena A Konovalova, Oleg S Gudyrev, Vladimir I Kochkarov, Liliya V Korokina, Eleonora N Dudina, Anna V Babko, Elena G Terehova Int J Hypertens 2011 ;2011 :515047).
[0012] Because of this, an intake of L-Arginine alone is not particularly effective in stimulating nitric oxide synthesis because of the action of Arginase in intestinal cells and blood vessel endothelial cells.
[0013] Other dietary supplements supporting nitric oxide production contain L -Citrulline. Originally identified in 1914 from the watermelon (Citrullus lanatus), from which it takes its name, L-citrulline is a non-essential, non-proteinogenic amino acid naturally present in the human body.
[0014] L-Citrulline is a precursor to L-Arginine and, differently to the latter, it is not subject to degradation by the enzyme Arginase, either in the intestine or in the endothelial cells of blood vessels. L-Citrulline therefore has a higher bioavailability than L-Arginine, as the body absorbs it more efficiently once taken orally. As it reaches the endothelial cells of blood vessels it can be converted to L-Arginine, thus participating in the synthesis of nitric oxide.
[0015] However, L-Citrulline alone cannot completely replace the functions of L-Arginine. Indeed, L-Citrulline has been shown to be more effective when administered together with L-Arginine. Noteworthy, the combination of the two amino acids in a 1 :1 ratio is more effective in stimulating nitric oxide synthesis than using each amino acid alone (Masahiko Morita, Toshio Hayashi, Masayuki Ochiai, Morihiko Maeda, Tomoe Yamaguchi, Koichiro Ina, Masafumi Kuzuya, Biochem Biophys Res Commun 2014 Nov 7;454(1 ):53-7). This is due to L-Citrulline ability to allosterically inhibit the enzyme Arginase both in the small intestine and in liver.
[0016] For this reason, many dietary supplements on the market within that range of products defined as "pre-workout” (i.e. to be taken before physical exertion to increase blood flow to muscle level and thus athletic performance) often contain a combination of L-Arginine and L-Citrulline.
[0017] L-Arginine and/or L-Citrulline are present in compositions having a vasodilator effect even in the presence of other formulations, such as in patent application n. RU2464019C1 , disclosing a composition having an endothelium-protective, vasodilator, and angio-protective effect, containing a NO donors such as L-arginine or L-citrulline and an Arginase inhibitor selected from L-Norvaline and nor-NONA.
[0018] L-Norvaline (a-aminovaleric acid) is a non-essential non-proteogenic amino acid which is an inhibitor of the enzyme Arginase in the human body (R. Rognstad, Biochim Biophys Acta 1977 Feb 28;496(2):249-54). It is slows down the ability of Arginase to capture Arginine within the urea cycle. L-Norvaline also inhibits the enzyme Ornithine
Transcarbamylase (OTC) further inhibiting the urea cycle (Hans-Peter Bode, Bernadette Eder, Michael Trautmann, European Journal of Biochemistry, June 1994222(3):69-877).
[0019] Some in vitro and in vivo evidence has, however, observed that the combination of L- Citrulline with Glutathione is able to elevate nitric oxide levels more than the single intake of L-Citrulline or Glutathione (Sarah McKinley-Barnard, Tom Andre, Masahiko Morita & Darryn S. Willoughby, Journal of the International Society of Sports Nutrition 2015 12, Article number: 27).
[0020] Glutathione is a tripeptide composed of the amino acids L-glutamate, L-cysteine, and glycine. Glutathione is an extremely potent antioxidant and detoxifying agent ubiquitously present in cells’ cytoplasm in the human body. It is able to inactivate the free radicals produced in the cells and to inhibit their ability to react and damage membranes and other biomolecules present in the cell, such as proteins and nucleic acids. Oral administration of Glutathione is effective in increasing the body's stores of Glutathione (John P Richie Jr, Sailendra Nichenametla, Wanda Neidig, Ana Calcagnotto, Jeremy S Haley, Todd D Schell, Joshua E Muscat, Eur J Nutr 2015 Mar;54(2):251 -63).
[0021] Studies have shown that Glutathione has the ability to upregulate the action of nitric oxide. Indeed, nitric oxide has a relatively short half-life and is subject to degradation by reaction with free radicals. Glutathione is able to deactivate free radicals, thus shielding and protecting nitric oxide from oxidative damage and enhancing the shelf-life and, thus, the effectiveness of nitric oxide (O Pechanova 1 , M Kashiba, M Inoue, Jpn J Pharmacol 1999 Oct;81 (2):223-9). In addition, upon interaction with nitric oxide, Glutathione forms S-nitrosoglutathione, stabilizing nitric oxide itself and further protecting it from free radicals attack. S-nitrosoglutathione is then able to release and liberate nitric oxide via reconversion to free glutathione and nitric oxide (Shigeru Oae & Kbichi Shinhama, The
New Journal for Organic Synthesis, Volume 15, 1983 - Issue 3). [0022] A composition containing L-Citru 11 i ne or a salt thereof and Glutathione or a salt thereof is the subject of patent application WO2018221654A1 for use in increasing the ratio of muscle mass to total body weight in combination with a resistance workout program.
[0023] It is important to emphasize that an improved ability to sustain athletic physical activity, whether professional or amateur, is important for sustaining health in terms of numerous metabolic functions as well as by counteracting sarcopenia. In addition, imbalances and/or insufficient endogenous nitric oxide synthesis is a risk factor for the development of endothelial dysfunction and the development of cardiovascular disease (Mihail V. Pokrovskiy, Mihail V. Korokin, Svetlana A. Tsepeleva, Tatyana G. Pokrovskaya, Vladimir V. Gureev, Elena A. Konovalova, Oleg S. Gudyrev, Vladimir I. Kochkarov, Liliya V. Korokina, Eleonora N. Dudina, Anna V. Babko, and Elena G. Terehova, Int J Hypertens. 2011 ; 2011 : 515047).
[0024] Even in the strictly medical field, the possibility to modulate (generally increase) nitric oxide production arouses much interest too. Many drugs that can trigger and/or prolong the effects of nitric oxide have been developed in recent decades. Citing the best known, Sildenafil citrate (active ingredient in Viagra®) has several applications: treatment of erectile dysfunction, treatment of pulmonary arterial hypertension, treatment of vasospasm and ischemia in subjects with secondary Raynaud's phenomenon. However, Sildenafil citrate, as well as all artificial substances with pharmacological activity, possesses side effects: dizziness, flushing, gastroesophageal reflux, headache, and dyspepsia among the most common (K E Andersson, Br J Pharmacol 2018 Jul;175(13):2554-2565).
Summary of the invention
[0025] The issue addressed by the present invention is to provide a new composition useful for stimulating nitric oxide production with molecules already present in nature and free of adverse side effects.
[0026] This issue is solved by the composition and its use as outlined in the attached claims, whose definitions are integral part of the present description.
[0027] In particular, the invention relates to a composition to be used as a vasodilator and/or in a method for increasing blood flow in skeletal muscle.
[0028] Further features and advantages relative to the use of the composition of the invention, such its medical use, will result from the description of the examples of realization of the invention, provided as an indication of the invention.
Brief figures descriptions
[0029] Figure 1 shows the kinetics of nitric oxide production in HMVEC cells (Human MicroVascular Endothelial Cells).
[0030] Based on the fluorescence intensity of the DAF-FM DA probe under the four conditions tested: “CN” (negative control: L-Arginine + L-Citrulline); “Norv” (L-Arginine + L-Citrulline + L-Norvaline); “GSH” (L-Arginine + L-Citrulline + Glutathione); “Norv GSH” (L-Arginine + L-Citrulline + L-Norvaline + Glutathione), as described in Example 1 . The black arrow indicates the addition of L-Norvaline and/or Glutathione, where applicable.
[0031] Figure 2 shows nitric oxide production calculated after the addition of L-Norvaline or Glutathione (where applicable) (at minute 60' of the experiment), i.e. from the time when L-Arginine L-Citrulline, L-Norvaline and Glutathione are simultaneously present in the “Norv GSH” condition.
[0032] Figure 3 illustrates the VAS (visual analogue scale) scores of the five questions of the questionnaire evaluating the combined effect of L-Arginine or a salt thereof, L-Citrulline or a salt thereof, Glutathione and L-Norvaline or a salt thereof for each group of amateur athletes following training after taking placebo or after taking one of the four treatment mixtures:
- Arg + Cit: oral supplementation with mixture consisting of L-Arginine L-Aspartate + L-Citrulline;
Norv: oral supplementation with mixture consisting of L-Norvaline;
GSH: oral supplementation with mixture consisting of Glutathione;
- Arg + Cit + Norv + GSH: oral supplementation with mixture consisting of L-Arginine L-Aspartate + L-Citrulline + L-Norvaline + Glutathione.
[0033] Figure 4 shows the change in VAS scores in each group of amateur sportsmen and sportswomen to the questions in the questionnaire assessing the combined effect of L- Arginine or a salt thereof, L-Citrulline or a salt thereof, Glutathione and L-Norvaline or a salt thereof after placebo intake and after intake of one of the four treatment mixtures: Arg + Cit mixture, Norv mixture, GSH mixture, Arg + Cit + Norv + GSH mixture.
Detailed description of the invention
[0034] An object of the present invention is a composition comprising L-Arginine or a salt thereof, L-Citrulline or a salt thereof, L-Norvaline or a salt thereof, and Glutathione.
[0035] Indeed, it has been surprisingly found that a composition comprising L-Arginine or a salt thereof, L-Citrulline or a salt thereof, L-Norvaline or a salt thereof, and Glutathione can indeed be effectively employed in medicine, as a medicament. This composition has in fact been found to be particularly effective in the treatment of the human or animal body as a vasodilator and/or in a method of increasing blood flow in skeletal muscle.
[0036] In other words, a composition comprising L-Arginine or a salt thereof, L-Citrulline or a salt thereof, L-Norvaline or a salt thereof and Glutathione is effective as a vasodilator and, therefore, can be effectively employed as a vasodilating agent and/or in a method to increase blood flow in skeletal muscle.
[0037] Thus, according to one form of realization of the present invention, a composition comprising L-Arginine or a salt thereof, L-Citrulline or a salt thereof, L-Norvaline or a salt thereof, and Glutathione for use as a vasodilator and/or in a method for increasing blood flow in skeletal muscle is preferred.
[0038] In the composition comprising L-Arginine or a salt thereof, L-Citrulline or a salt thereof, L-Norvaline or a salt thereof and Glutathione, the salt of L-Arginine is chosen from: L- Arginine Hydrochloride, L-Arginine a-Ketoglutarate, L-Arginine Pyroglutamate, L- Arginine L-Aspartate, Di-L-Arginine Malate.
[0039] In the composition comprising L-Arginine or a salt thereof, L-Citrulline or a salt thereof, L-Norvaline or a salt thereof and Glutathione, the salt of L-Citrulline is L-Citrulline Malate.
[0040] In the composition comprising L-Arginine or a salt thereof, L-Citrulline or a salt thereof, L-Norvaline or a salt thereof, and Glutathione, the amount of L-Arginine or a salt thereof is from 0.1 to 5 grams, the amount of L-Citrulline or a salt thereof is from 0.1 to 5 grams, the amount of Glutathione is from 0.01 to 0.50 grams and the amount of L-Norvaline or a salt thereof is from 0.1 to 5 grams, per dose.
[0041] In the composition comprising L-Arginine or a salt thereof, L-Citrulline or a salt thereof, L-Norvaline or a salt thereof and Glutathione, the percentage of L-Arginine or a salt thereof is from 0.65 to 45.0 % (w/w), the percentage of L-Citrulline or a salt thereof is from 0.65 to 50.0 % (w/w), the percentage of Glutathione is from 0.065 to 20.0 % (w/w) and the percentage of L-Norvaline or a salt thereof is from 0.65 to 35.0 % (w/w).
[0042] It is another object of the invention to provide a food preparation or beverage comprising a composition including L-Arginine or a salt thereof, L-Citrulline or a salt thereof, L- Norvaline or a salt thereof, and Glutathione, including the preferred forms of realization described above.
[0043] It is another object of the invention to provide a food supplement comprising a composition including L-Arginine or a salt thereof, L-Citrulline or a salt thereof, L- Norvaline or a salt thereof, and Glutathione, including the preferred forms of implementation described above, in powder or tablet or capsule or soft capsule or liquid or gel or bar form.
[0044] It is another object of the invention to provide a pharmaceutical composition comprising L-Arginine or a salt thereof, L-Citrulline or a salt thereof, L-Norvaline or a salt thereof, and Glutathione, including the preferred forms of realization described above, and at least a pharmaceutically acceptable excipient.
[0045] Such pharmaceutical composition may be in solid form, i.e. in tablet or capsule form, or in liquid form, i.e. in the form of an injectable solution or syrup.
[0046] The pharmaceutical composition comprising L-Arginine or a salt thereof, L-Citrulline or a salt thereof, L-Norvaline or a salt thereof, and Glutathione, including the preferred realizations described above, and at least a pharmaceutically acceptable excipient is to increase blood flow in skeletal muscle.
[0047] Another object is the composition described above, including the various embodiments, for use in medicine.
[0048] In particular, another object is the aforementioned composition for use in a method of treating the human or animal body as a vasodilator and / or in a method for increasing blood circulation in the skeletal muscle.
[0049] Finally, a further object is the use of the composition described above as a food supplement.
[0050] The following table (I) gives evidence of the effects provided by the composition according to the present invention (Leibovitz Norv GSH group) as a vasodilating agent and/or in a method for increasing blood flow in skeletal muscle.
[0051] Table I
Figure imgf000012_0001
[0052] The values in table (I) were acquired according to the methods and conditions described in Example 1 .
[0053] Table (I) indicates that there is no difference between CN group (negative control; observed combination: L-Arginine + L-Citrulline) and Norv group (observed combination: L-Arginine + L-Citrulline + L-Norvaline), while the fluorescence of the nitric oxide probe in the simultaneous presence of L-Arginine, L-Citrulline, glutathione and L-Norvaline (condition 'Leibovitz Norv GSH') is statistically significant (p < 0.0001 ) compared to all other three conditions.
[0054] Furthermore, it can be seen from Figures 1 and 2 as well as in Table (I) that in the 'Leibovitz Norv GSH' condition (co-presence of L-Arginine + L-Citrulline + L-Norvaline + Glutathione) a synergy can be observed among the 4 substances used in this condition, as the increase in nitric oxide over the course of the experiment is greater than the sum of the increases in the other conditions, i.e. of the substances administered individually or in pairs. This indicates a synergy among L-Arginine, L-Citrulline, L-Norvaline and
Glutathione that has not been previously shown in the scientific literature.
[0055] Tests performed in athletes confirmed what had been seen in the experiments in HMVEC cells, as participants perceived a higher improvement in performance in the 'Arg + Cit + Norv + GSH' group than in groups in which these substances were taken individually or in pairs.
[0056] EXPERIMENTAL PART
[0057] Evaluation of the combined effect of L-Arginine or a salt thereof, L-Citrulline or a salt thereof, Glutathione and L-Norvaline or a salt thereof was performed in:
In vitro: immortalized cell cultures of HMVEC cells;
In vivo: amateur athletes.
[0058] Example 1 : Evaluation of the combined effect of L-Arginine or a salt thereof, L-Citrulline or a salt thereof, Glutathione and L-Norvaline or a salt thereof in immortalized Human MicroVascular Endothelial Cells (HMVEC) cultures.
[0059] HMVEC cells were chosen because they are one of the main sites of nitric oxide production in the body.
[0060] HMVEC cells were grown in M199 culture medium supplemented with FBS and growth factors and loaded into Leibovitz L-15 culture medium with DAF-FM DA probe (abeam: ab145295) 15 pM for 15'. The DAF-FM DA probe is internalized by the cells and, once inside, is able to react with nitric oxide to produce a fluorescent compound. Fluorescence emission is therefore proportional to the nitric oxide produced in the cell.
[0061] After loading the nitric oxide-sensitive DAF-FM DA probe, appropriate cell washes were performed to remove excess DAF-FM DA from the extracellular culture medium.
[0062] The following four conditions were tested : [0063] CN (negative control): HMVEC cells maintained in Leibovitz culture medium with L- Arginine 2mM + L-Citrulline 2mM for 1 h. Combination observed: L-Arginine + L-Citrulline.
[0064] Norv: HMVEC cells maintained in Leibovitz culture medium with L-Arginine 2mM + L- Citrulline 2mM for 1 h. After 30', L-Norvaline 2mM was added for a further 30'. Combination observed: L-Arginine + L-Citrulline + L-Norvaline.
[0065] GSH: HMVEC cells maintained in Leibovitz culture medium with L-Arginine 2mM + L- Citrulline 2mM for 1 h. After 30', Glutathione 2mM was added for a further 30'. Combination observed: L-Arginine + L-Citrulline + Glutathione.
[0066] Norv GSH: HMVEC cells maintained in Leibovitz culture medium with L-Arginine 2mM + L-Citrulline 2mM for 1 h. After 30', L-Norvaline 2mM + Glutathione 2mM was added for a further 30'. Combination observed: L-Arginine + L-Citrulline + L-Norvaline + Glutathione.
[0067] For each condition, a fluorescence measurement was performed in the cells every 5'. Each condition was tested in triplicate and the total number of cells for each condition is greater than or equal to n=47. Means ± Standard Errors of each condition are shown in Figure 1 .
[0068] In order to assess the statistical significance of the results obtained, One Way ANOVA test was performed. The generated p-values were corrected with Sidak's correction for multiple testing. The test results are summarised in table (I).
[0069] Example 2: Evaluation of the combined effect of L-Arginine or a salt thereof, L-Citrulline or a salt thereof, Glutathione and L-Norvaline or a salt thereof in amateur athletes.
[0070] Participants recruitment: subjects participating in the trial evaluating the efficacy of the invention were selected as follows: healthy subjects, of both sexes, aged between 20 and 55 years, who habitually practiced (at least twice a week) amateur or professional sports activities lasting between 1 h and 2h, and falling within the following disciplines: body building, gym fitness classes, crossFit®, calisthenics, martial arts and combat sports, team sports (football, futsal, volleyball, rugby, basketball).
[0071] A total of 31 people participated in the study, 12 women and 19 men.
[0072] Each participating subject was informed about the purpose of the test and consented to his/her participation.
[0073] All ingredients used in the mixtures taken by the participants are substances with a nutritional effect or excipients permitted by Italian Ministry of Health for use in food supplements.
[0074] Study protocol: the study was double-blind with internal control.
[0075] Each participating subject was required to take a powder mixture dissolved in water (100- 250 mL) 45'-60' before training for two workouts. Following each workout, each participant was asked to complete an evaluation questionnaire.
[0076] Each participant received, in one training session, a 'treatment' mixture containing one or more of the substances L-Arginine or a salt thereof, L-Citrulline or a salt thereof, L- Norvaline or a salt thereof and Glutathione and, in another training session, a 'placebo' mixture containing none of the above substances but with an appearance, smell and taste similar on the ‘treatment’ mixture.
[0077] Neither the study participants nor the investigators were aware of which was the ‘treatment’ mixture and which was the placebo mixture at the time of the training.
[0078] The treatment mixtures are as follows:
- Arg + Cit: containing L-Arginine L-Aspartate, L-Citrulline;
Norv: containing L-Norvaline;
GSH: containing Glutathione;
Arg + Cit + Norv + GSH: containing L-Arginine L-Aspartate, L-Citrulline, L-Norvaline and Glutathione.
[0079] Evaluation questionnaire. The evaluation questionnaire completed by the study participants after training is as follows.
Did you warm up quickly at the start of the training?
Did you feel energetic to perform your effort?
Did you feel muscle pain the day after training?
Do you think the use of the mixture has improved your performance?
- Would you reuse the mixture you have tried?
[0080] Data were collected by means of VAS (Visual Analogue Scale) score where 0 stands for the minimum value that can be given to the question asked ('not at all agree', 'not at all often'), and 10 stands for the maximum value ('absolutely agree', 'always').
[0081] Data analysis: The data obtained were analysed using Prism (GraphPad Software,
USA).

Claims

Claims
1 ) Composition comprising L-Arginine or a salt thereof, L-Citrulline or a salt thereof, L- Norvaline or a salt thereof and Glutathione.
2) Composition according to claim 1 , wherein the L-Arginine salt is selected from: L-arginine hydrochloride, L-Arginine a-ketoglutarate, L-Arginine Pyroglutamate, L-Arginine L- Aspartate and Di-L-Arginine Malate.
3) Composition according to claim 1 , wherein the L -Citrulline salt is L-Citrulline Malate.
4) Composition according to any one of claims 1 to 3, wherein the percentage of L-Arginine or a salt thereof is from 0.65 to 45.0 % (w/w), the percentage of L-citrul I i ne or a salt thereof is from 0.65 to 50.0 % (w/w), the percentage of Glutathione is from 0.065 to 20.0 % (w/w) and the percentage of L-Norvaline or a salt thereof is from 0.65 to 35.0 % (w/w).
5) Composition according to any one of claims 1 to 3, wherein the amount of L-Arginine or a salt thereof is from 0.1 to 5 grams, the amount of L-citrulline or a salt thereof is from 0.1 to 5 grams, the amount of Glutathione is from 0.01 to 0.50 grams and the amount of L- Norvaline or a salt thereof is from 0.1 to 5 grams, per dose.
6) Food or beverage preparation comprising a composition according to any one of claims 1 to 5.
7) Food supplement comprising a composition according to any one of claims 1 to 5, in the form of powder or tablet or capsule or soft capsule or liquid or gel or bar.
8) Pharmaceutical composition comprising a composition according to any one of claims 1 to 5 and at least one pharmaceutically acceptable excipient.
9) Composition according to any one of claims 1 to 5, for use in medicine. ) Composition according to any one of claims 1 to 5, for use in a method of treatment of the human or animal body as a vasodilator and / or in a method to increase blood flow in skeletal muscle. ) Use of the composition according to any one of claims 1 to 5 as a food supplement.
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