Novel formulations of glutathione

Abstract

The present disclosure provides novel formulations for biologically active molecules including glutathione and functional analogs of glutathione. These formulations comprise magnesium stearate or a salt thereof with comparable properties along with a phospholipid carrier complex. These formulations provide for more convenient modes of delivery, such as via orally consumed capsules containing a solid powder to either human or animal subjects.

Description

PATENTAttorney Docket No. 115653-1526569-000410WONOVEL FORMULATIONS OF GLUTATHIONECROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 715,633, filed November 3, 2024, which application is incorporated herein by reference in its entirety for all purposes.FIELD

[0002] The invention relates to new methods of formulation of phospholipid carrier complexes and uses thereof to enable novel modes of delivery of biologically active molecules such as glutathione.BACKGROUND

[0003] Glutathione is a sulfur-bearing nitrogenated organic molecule that functions as an antioxidant across biological Kingdoms. Chemically, it is a tripeptide, comprising three amino acids, with a gamma peptide linkage between the carboxyl group of the glutamate side chain and cysteine. In addition to its cellular protectant properties, glutathione has been found to have anti-melanogenic properties and has been promoted as a skin-lightening agent (Sonthalia et al., 2016, Indian J Dermatol. Venereol. Leprol. 82(3):262-272. Glutathione has also been proposed to enhance sleep quality and / or reduce anxiety since it protects against cellular oxidative stress, which is associated with maladies such as sleep disturbance and chronic stress.

[0004] Orally ingested glutathione has poor bioavailability because the tripeptide is the substrate of peptidases of the alimentary canal as well as the absence of a specific carrier of glutathione in the cell membrane (Witschi et al., 1992, European Journal of Clinical Pharmacology. 43 (6): 667-669). Novel improved formulations for delivery of the molecule to human or animal subjects which provide for improved bioavailability are disclosed herein.SUMMARY

[0005] The disclosed processes can be applied for use to produce formulated health supplement products or prescription drugs. The use of magnesium stearate and / or a salt with comparable properties as a component within formulations comprising glutathione or afunctional analog and a phospholipid carrier complex is disclosed. The inclusion of magnesium stearate or a comparable salt offers important improvements over the existing art and enables the delivery of these molecules in solid form (for example in tablet or capsule), such that they can be delivered orally. A particular example comprises a formulation containing glutathione, a phospholipid carrier complex, and magnesium stearate, provided as capsule based oral formulation. This method of delivery is superior to the alternative modes of delivery, such as injection of a liquid formulation.SPECIFIC EMBODIMENTS

[0006] Glutathione is typically delivered in liquid formulations, which are consumed orally, but the shelf life of these formulations is poor and subjects report that the oral formulations have an unpleasant taste. As an alternative, liquid formulations of glutathione can be delivered by injection or by “snorting” nasally but subjects report these methods to be extremely painful. As a further alternative, the liquid formulations can be delivered intravenously, but this method of dosing is inconvenient as it requires the patient to visit a medical facility and the procedure may take several hours. Thus, there is a real-world need for improved formulations of glutathione. Improved formulations that can be delivered orally or via a variety of other application methods are disclosed herein.

[0007] Liposomes are closed bilayer structures spontaneously formed by hydrated phospholipids that are widely used as efficient delivery systems for drugs or antigens, due to their capability to encapsulate bioactive hydrophilic, amphipathic, and lipophilic molecules into inner water phase or within lipid leaflets (Nisini et al., 2018, Front. Immunol., Sec. Vaccines Molec. Therapeutics 9: 155; also reviewed by Liu et al. (2022). Molecules. 27: 1372. Phospholipid carrier complexes are a class of liposomal product which have various applications in drug formulation including to enhance bioavailability of drugs with low aqueous solubility or low membrane penetration, improvement of the uptake and release profile of drugs, protection of sensitive bioactive agents from degradation in the gastrointestinal tract, reduction of side effects and masking of the bitter taste of drugs. Phospholipids have the exceptional biocompatibility and remarkable amphiphilicity characteristics that make them suitable agents to achieve better therapeutic applications in many drug-delivery systems (Singh et al. (2017), J. Drug Delivery Sci. Technol. 39: 166-

[0008] Phospholipid carrier complexes have been used with various bioactive chemicals for many years, as have other methods of making liposomes. These liposomal products can be divided into two types of formulation: the very simple, typically based on 1-3 ingredients which mostly occur in nature, versus the very complex which contain purely synthetic components (for example mRNA-based vaccines). Typically, the simple methods of formulation involve the inclusion of lecithin or phosphatidylcholine (one of the main components of lecithin) or bile salts as the bulk of the liposomal carrier. The problems with these simple formulation methods are that (a) if used in powder form, lecithin and phosphatidylcholine are hygroscopic and quickly become very sticky and unusable, and (b) if used in liquid form, a substantial quantity of antimicrobial preservative and / or antioxidant is required to prevent oxidation of the liposomal carrier ingredient as well as oxidation of the active ingredient and / or the growth of microbes. In the latter applications, since the liposomal formulation acts to make molecules more bioavailable, the preservative compounds also become more bioavailable, which is undesirable and can cause side effects in the subject.

[0009] The novel formulation method disclosed herein involves the addition of a significant quantity (over 10-20% by weight) of magnesium stearate (or a salt with comparable properties) as a hydrophobic component, to prevent the absorption of water from the air or surrounding environment during storage. The inclusion of a hydrophobic component like magnesium stearate in such formulations prevents them from becoming sticky and maintains them in a usable powder form.

[0010] Additionally, the addition of magnesium stearate or comparable agent into the formulation may facilitate the rapid dispersion of the phosphatidylcholine / chemical complex once it enters an aqueous environment (in the intestines) leading to more rapid absorption of the liposomal complex and thereby achieving an increased efficacy of the bioactive ingredient in the human or animal subject. The methodologies herein encompass novel uses and delivery methods for glutathione.DEFINITIONS:

[0011] “AUC” means Area Under Curve, a standard measurement made in pharmacokinetics which indicates the concentration of a drug in the bloodstream versus the time since dosing. In practice, the drug concentration is measured at certain discrete points in time and the trapezoidal rule is used to estimate AUC. In pharmacology, the area under theplot of plasma concentration of a drug versus time after dosage indicates the extent of exposure to a drug and its clearance rate from the body.

[0012] “Functional analog” means, with respect to a first chemical molecule active ingredient, a second chemical molecule that possess a similar structure to the first molecule such as a carbon backbone with common features, and which has similar biological activity.

[0013] “ Glutathione” means the molecule with the chemical formula chemical formula HOCOCH(NH2)CH2CH2CONHCH(CH2SH)CONHCH2COOH and the systematic IUPAC gamma-Glutamylcysteinylglycine. It is also known as (2S)-2-Amino-5-({(2R)-l- [(carboxymethyl)amino]-l-oxo-3-sulfanylpropan-2-yl}amino)-5-oxopentanoic acid as well as gamma-L-Glutamyl-L-cysteinylglycine and (2S)-2-Amino-4-({(lR)-l [(carboxymethyl)carbamoyl]-2-sulfanylethyl}carbamoyl)butanoic acid.

[0014] “Inflammatory immune response” is a normal function of the immune system in response to tissue injury, the body initiates a chemical signaling cascade that stimulates responses aimed at healing affected tissues. These signals activate leukocyte chemotaxis from the general circulation to sites of damage. These activated leukocytes produce cytokines that induce inflammatory responses. In chronic diseased states this inflammatory immune response can cause systemic deleterious effects on the body.

[0015] “Lecithin” or “lecithins” refer to one or more fatty substance(s) occurring in, or derived from, animal or plant tissues, which shows amphiphilic properties; that is, they attract both water and fatty substances (and thus are both hydrophilic and lipophilic). Lecithins are mixtures that include glycerophospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, and / or phosphatidic acid. Lecithin can readily be extracted chemically using organic chemical solvents or extraction can be done mechanically. Common substrates from which lecithin is extracted include egg yolk, seafood, soybeans, milk, rapeseed, cottonseed, and sunflower oil. Lecithin has low solubility in water, but can be included in drug formulations to fulfil the role of a liposomal carrier complex. In aqueous solution, the phospholipids of lecithin can form either liposomes, bilayer sheets, micelles, or lamellar structures, depending on hydration and temperature.

[0016] “Liposome” refers to a self-assembled (phospho)lipid-based drug vesicle that forms a bilayer (unilamellar) and / or a concentric series of multiple bilayers (multilamellar) enclosing a central compartment, which may contain a drug molecule.

[0017] “Liposomal carrier complex” or “phospholipid carrier complex” refers to a liposome to which additional chemical components have been added to reduce biodegradation and enhance the efficiency of delivery. Liposomal carrier complexes may incorporate an amphiphilic substance such as lecithin or a polymer, which can increase the stability of liposome with regard to pH, chemicals, enzymes, and the immune system.

[0018] “Melasma” refers to a tan or dark skin discoloration. Melasma is thought to be caused by sun exposure, genetic predisposition, hormone changes, and skin irritation. In some instances, the term is used to describe a skin condition whereby dark patches and spots appear, particular on the face, which is sometimes associated with pregnancy or the use of birth control pills.

[0019] “Magnesium stearate” means the salt with the chemical formula C36H70MgO4 containing two equivalents of stearate (the anion of stearic acid, an 18-carbon carboxylic acid) and one magnesium cation (Mg+2). Magnesium stearate is a white, water-insoluble powder. Its applications typically exploit its insolubility or low solubility in many solvents, and low toxicity. Salts comprising Mg2+ or another divalent cation combined with the anion of stearate or the anion of another carboxylic acid with a similar length carbon chain may be expected to exhibit similar physical properties.

[0020] “Physical properties” means, with respect to a chemical, features such as its melting point and solubility.EXAMPLESEXAMPLE 1. Inclusion of magnesium stearate in a formulation containing glutathione.

[0021] A formulation was prepared comprising magnesium stearate at rate of at least 10g, or at least 11g or at least 12g, or at least 13g, or at least 14g or at least 15g or at least, or at least 16g or at least, or at least 17g or at least, or at least 18g, or at least 19g, or at least 20g per 100g of total formulation, which also contained glutathione (at least 2g and up to 80g per 100g of total formulation) and lecithin (at least 2g and up to 88g per 100g of total formulation of which at least 30% consists of phospholipids). The formulation was visibly finer and less prone to clumping and agglomeration compared to a control formulation lacking magnesium stearate, after 7 days of leaving it exposed to air. It was observed that the addition of magnesium stearate dramatically changed the physical characteristics of the formulation; without the magnesium stearate, the control formulation agglomerated into sticky mass. The formulation with the magnesium stearate present remained in the form of a very fine powder.EXAMPLE 2. Inclusion of magnesium stearate in a formulation containing glutathione and phosphatidylcholine.

[0022] A formulation was prepared comprising magnesium stearate at rate of at least 10g, or at least 11g or at least 12g, or at least 13g, or at least 14g or at least 15g or at least, or at least 16g or at least, or at least 17g or at least, or at least 18g, or at least 19g, or at least 20g per 100g of total formulation, which also contained glutathione (at least 2g and up to 80g per 100g of total formulation) and phosphatidylcholine (at least 2g and up to 67g per 100g of total formulation). The formulation was visibly finer and less prone to clumping and agglomeration compared to a control formulation lacking magnesium stearate, after 5 days of leaving it exposed to air. It was observed that the addition of magnesium stearate dramatically changed the physical characteristics of the formulation; without the magnesium stearate, the control formulation agglomerated into sticky mass. The formulation with the magnesium stearate present, remained in the form of a very fine powder.EXAMPLE 3. A simple dispersibility test to model the dispersion of a drug within the body of a subject.

[0023] The following method can be used. Stir a given amount of powdered formulation in distilled water at 37 degrees Centigrade. Allow the suspension to settle for a period of 5 minutes at and visibly observe the amount of suspended material versus sediment.Alternatively, the amount of suspended material may be quantified by an IR refraction-based method.

[0024] The formulations described in examples 1-4 were suspended in water according to the above method. In each instance, a visibly greater quantity of the formulation remained in the aqueous fraction after a five-minute period in the formulations containing magnesium stearate, as compared to the control formulations. Based on these results, it was concluded that the addition of magnesium stearate likely enhances the bioavailability of the active ingredients within a human or animal subject.EXAMPLE 4. A formulation comprising glutathione for the lightening of skin tone.

[0025] A novel formulation is provided to subjects that includes glutathione (at least 2g and up to 80g per 100g of total formulation) in combination with either bile salts (at least 2g and up to 67g per 100g of total formulation), phosphatidylcholine (at least 2g and up to 67g per 100g of total formulation) and / or lecithin (at least 2g and up to 86g per 100g of total formulation of which at least 30% consists of phospholipids) in combination with magnesium stearate at rate of at least 10g, or at least 11g or at least 12g, or at least 13g, or at least 14g orat least 15g or at least, or at least 16g or at least, or at least 17g or at least, or at least 18g, or at least 19g, or at least 20g per 100g of total formulation. The formulation is provided to one or more subjects and control formulations lacking one or more components are provided to one or more subjects as part of an observational study. The formulation is concluded to have a benefit in lightening skin coloration.EXAMPLE 5. A formulation comprising glutathione in combination with estrogen or progesterone for the amelioration of the effects of melasma associated with the use of birth control pills.

[0026] A novel formulation is provided to subjects which includes glutathione (at least 2g and up to 80g per 100g of total formulation) in combination with either bile salts, (at least 2g and up to 67g per 100g of total formulation), phosphatidylcholine (at least 2g and up to 67g per 100g of total formulation) and / or lecithin (at least 2g and up to 86g per 100g of total formulation of which at least 30% consists of phospholipids) in combination with magnesium stearate at rate of at least 10g, or at least 11g or at least 12g, or at least 13g, or at least 14g or at least 15g or at least, or at least 16g or at least, or at least 17g or at least, or at least 18g, or at least 19g, or at least 20g per 100g of total formulation, and in combination with a sex hormone such as estrogen or progesterone or an analog thereof. The formulation is provided to one or more subjects and control formulations lacking one or more components are provided to one or more subjects, as part of an observational study. The formulation is concluded to have a benefit in lightening skin coloration and / or reducing the symptoms of melasma.EXAMPLE 6. Assessment of the availability of glutathione administered according to examples 1 or 3 above by oral or sublingual route.

[0027] AUC blood testing is performed by means of measuring total glutathione via a standard blood test that is readily available clinically.EXAMPLE 7. New products based on novel formulations and specific use cases.

[0028] At least two specific use cases exist of the methods disclosed herein for increasing absorption and bioavailability of bioactive chemicals. These are listed as (a)-(b) below:(a) to increase the AUC of glutathione in the bloodstream. The specific use of this product is to improve sleep quality or duration in humans, to inhibit the inflammatory immune response in humans and / or animals and / or make the skin coloration lighter in tone and / or to improve conditions of skin discoloration caused by melanin, such as melasma, and / or to reduce tanning;(b) to increase the absorption of glutathione into intestinal epithelial cells.EXAMPLE 8.

[0029] The present disclosure is directed in part to chemical formulations that comprise a glutathione or a functional analog, and a salt or salts that function in a manner comparable to magnesium stearate, that is, a salt or salts with comparable properties to magnesium stearate. The salt or salts with said comparable properties may be selected from the group consisting of magnesium stearate, magnesium laurate, magnesium palmitate, magnesium myristate, calcium stearate, calcium laurate, calcium palmitate, calcium myristate, sodium stearate, sodium laurate, sodium palmitate, sodium myristate, sodium stearyl fumarate, zinc stearate, zinc laurate, zinc palmitate, zinc myristate, stearic acid, lauric acid, palmitic acid, and myristic acid.

[0030] Several of the salts in this list are commonly used for pharmaceuticals including magnesium stearate, stearic acid, and sodium stearyl fumarate.

[0031] Formulations of this disclosure may comprise:

[0032] Glutathione combined with magnesium stearate or a salt with comparable properties to magnesium stearate, including concentration ranges of:

[0033] magnesium stearate or a salt with comparable properties to magnesium stearate at a concentration of at least 5%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% w / w; or about 5%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% w / w, or in a range of 5% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, or 90% to 96%, or

[0034] glutathione or a functional analog at a concentration of at least 2g, 3g, 4g, 5g, 6g, 7g, 8g, 9g, 10g, 11g, 12g, 13g, 14g, 15g, 16g, 17g, 18g, 19g, 20g, 21g, 22g, 23g, 24g, 25g, 26g, 27g, 28g, 29g, 30g, 31g, 32g, 33g, 34g, 35g, 36g, 37g, 38g, 39g, 40g, 41g, 42g, 43g,44g, 45g, 46g, 47g, 48g, 49g, 50g, 51g, 52g, 53g, 54g, 55g, 56g, 57g, 58g, 59g, 60g, 61g,62g, 63g, 64g, 65g, 66g, 67g, 68g, 69g, 70g, 71g, 72g, 73g, 74g, 75g, 76g, 77g, 78g, 79g, or80g per 100g of total formulation, or in a range of 2g to 5g, 5g to 10g, 10g to 20g, 20g to 30g,30g to 40g, 40g to 50g, 50g to 60g, 60g to 70g, or 70g to 80g per 100g of total formulation; or

[0035] lecithin at a concentration of at least 2g, 3g, 4g, 5g, 6g, 7g, 8g, 9g, 10g, 11g, 12g, 13g, 14g, 15g, 16g, 17g, 18g, 19g, 20g, 21g, 22g, 23g, 24g, 25g, 26g, 27g, 28g, 29g, 30g,31g, 32g, 33g, 34g, 35g, 36g, 37g, 38g, 39g, 40g, 41g, 42g, 43g, 44g, 45g, 46g, 47g, 48g,49g, 50g, 51g, 52g, 53g, 54g, 55g, 56g, 57g, 58g, 59g, 60g, 61g, 62g, 63g, 64g, 65g, 66g,67g, 68g, 69g, 70g, 71g, 72g, 73g, 74g, 75g, 76g, 77g, 78g, 79g, 80g, 81g, 82g, 83g, 84g,85g, 86g, 87g, or 88g per 100g of total formulation, or in a range of 2g to 5g, 5g to 10g, 10g to 20g, 20g to 30g, 30g to 40g, 40g to 50g, 50g to 60g, 60g to 70g, 70g to 80g, or 80g to 88g per 100g of which at least 30% consists of phospholipids.EXAMPLE 9. A method of increasing absorption and bioavailability of a bioactive chemical to increase the AUC of the bioactive chemical in the bloodstream.

[0036] The method comprising the steps of combining 10% - 60% by weight of magnesium stearate in a formulation with lecithin, phosphatidylcholine, and / or bile salts in a formulation combination with the bioactive molecule and delivering said formulation to a human or animal subject by oral delivery or by injection.EXAMPLE 10.

[0037] The method of Example 8 wherein the biochemical active in the formulation is glutathione or a functional analog of glutathione that improves sleep quality or duration, and / or inhibits the inflammatory immune response in a humans and / or produces lighter skin coloration or reduces conditions of skin discoloration caused by melanin, such as melasma.EXAMPLE 11.

[0038] The method of Example 9, wherein the formulation increases the absorption of glutathione or a functional analog of glutathione into intestinal epithelial cells to reduce the generic symptoms of inflammatory disorders and / or lighten skin coloration.EXAMPLE 12.

[0039] The method of Example 10, wherein the formulation increases the AUC of glutathione or a functional analog of glutathione in the bloodstream.

Claims

WHAT IS CLAIMED IS:

1. A chemical formulation comprising glutathione or a functional analog of glutathione combined with magnesium stearate or a salt with comparable physical properties to magnesium stearate.

2. The chemical formulation of claim 1, wherein the chemical formulation also contains a liposomal carrier complex.

3. The chemical formulation according to claim 1, wherein the chemical formulation is an orally delivered drug.

4. The chemical formulation according to claim 1 or claim 2, wherein the chemical formulation is a drug that is delivered nasally, by skin application, subcutaneously or as a suppository.

5. The chemical formulation according to claim 1 or claim 2, wherein the chemical formulation comprises a solid dry powder.

6. The chemical formulation of claim 1 or claim 2, wherein the chemical formulation contains estrogen, an estrogen analog, progesterone, a progesterone analog, or a molecule that functions as a sex hormone.

7. The chemical formulation according to claim 1, wherein the chemical formulation contains magnesium stearate at a concentration of at least about 5%, 10%, 11%, 12%, 13%, 14%. 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% w / w.

8. The chemical formulation according to claim 1, wherein the chemical formulation is used to treat an inflammatory immune response, improve sleep quality / duration or to make skin lighter in coloration or to treat conditions of skin discoloration caused by skin discoloration or melasma.

9. A chemical formulation used as a birth control drug that includes glutathione or a functional analog of glutathione in the chemical formulation in combination with a molecule that functions as a sex hormone.

10. A method of treating inflammation improving sleep quality, improving sleep duration, lightening skin color, or treating conditions of skin discoloration caused by anexcess of, or an uneven distribution of melanin or melasma, comprising dosing a human or animal subject patient with a drug comprising a chemical formulation containing glutathione or a functional analog of glutathione in combination with magnesium stearate or a salt with comparable properties to magnesium stearate.

11. The method of claim 10, wherein the chemical formulation also contains a liposomal carrier complex.

12. The method according to either claim 10, wherein the chemical formulation is delivered orally, nasally, by skin application, intravenously, subcutaneously or as a suppository.

13. The method according to claim 10, wherein the formulation comprises a solid powder.

14. The method according to claim 10 wherein the formulation also contains a liposomal carrier complex.

15. The method according to claim 10, wherein the formulation contains magnesium stearate at a concentration of at least about 5%, 10%, 11%, 12%, 13%, 14%. 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% w / w.

16. A method for increasing the Area Under Curve (AUC) of a biologically active glutathione-like molecule comprising glutathione or a functional analog of glutathione, the method comprising combining magnesium stearate, or salt with comparable physical properties to magnesium stearate, into a formulation containing the glutathione-like molecule, and delivering the formulation to a human or animal subject, whereby the AUC is increased as compared to a subject treated with a control formulation lacking the magnesium stearate or its comparable salt.

17. The method according to claim 16, wherein the delivering of the drug is by oral, nasal, intravenous, subcutaneous, by skin application, or by suppository administration.

18. The method according to claim 16, wherein the formulation is a solid powder.

19. The method according to claim 16, wherein the formulation contains magnesium stearate or a salt with comparable properties at a concentration of at least about: 5%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, or 25% w / w.