WO2018045335A1 - Donneurs d'oxyde d'azote et leurs utilisations - Google Patents

Donneurs d'oxyde d'azote et leurs utilisations Download PDF

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Publication number
WO2018045335A1
WO2018045335A1 PCT/US2017/049946 US2017049946W WO2018045335A1 WO 2018045335 A1 WO2018045335 A1 WO 2018045335A1 US 2017049946 W US2017049946 W US 2017049946W WO 2018045335 A1 WO2018045335 A1 WO 2018045335A1
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composition
leucine
day
donors
subject
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PCT/US2017/049946
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English (en)
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Michael ZEMEL
Antje BRUCKBAUER
Jheelam BANERJEE
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Nusirt Sciences, Inc.
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Publication of WO2018045335A1 publication Critical patent/WO2018045335A1/fr

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    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • 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
    • A61K31/04—Nitro compounds
    • 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
    • A61K31/13—Amines
    • A61K31/155—Amidines (), e.g. guanidine (H2N—C(=NH)—NH2), isourea (N=C(OH)—NH2), isothiourea (—N=C(SH)—NH2)
    • 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
    • A61K31/196—Carboxylic acids, e.g. valproic acid having an amino group the amino group being directly attached to a ring, e.g. anthranilic acid, mefenamic acid, diclofenac, chlorambucil
    • 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
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/401—Proline; Derivatives thereof, e.g. captopril
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/455—Nicotinic acids, e.g. niacin; Derivatives thereof, e.g. esters, amides

Definitions

  • Hepatic steatosis also sometimes referred to as fatty liver disease, is a condition generally characterized by an abnormal retention of lipids in cells of the liver. Hepatic steatosis affects millions of people worldwide. The prevalence of fatty liver disease has been estimated to range from 10-24% in various countries around the globe. Fatty liver disease can be classified into several categories. For example, non-alcoholic fatty liver disease (NAFLD) generally refers to a spectrum of hepatic lipid disorders characterized by hepatic steatosis with no known secondary cause.
  • NAFLD non-alcoholic fatty liver disease
  • NAFLD can be subcategorized into (a) non-alcoholic fatty liver (NAFL), defined as the presence of steatosis in the absence of histological evidence of hepatocellular injury, and (b) non-alcoholic steatohepatitis (NASH), hepatic steatosis accompanied by hepatocyte injury and inflammation.
  • NASH may occur with or without fibrosis, but may progress to fibrosis and cirrhosis.
  • NAFLD is generally associated with energy metabolism pathologies, including obesity, dyslipidemia, diabetes and metabolic syndrome. The prevalence of NAFLD in the general population is estimated at 20%, with prevalence of NASH estimated to be 3-5%. Among patients with obesity (or diabetes) and patients with dyslipidemias, the prevalence rate of NAFLD is estimated 70% and 50%, respectively.
  • the present disclosure generally relates to regulation of fat accumulation in cells and/or tissue.
  • the present disclosure involves compositions, methods, and kits for reducing, treating, preventing, or sustaining the reduction of non-alcoholic steatohepatitis or hepatic steatosis in a subject in need thereof.
  • compositions comprising: a) leucine and/or a metabolite of leucine; and b) one or more NO (nitric oxide) donors.
  • composition comprising: a) an effective amount of leucine and/or an effective amount of a metabolite of leucine; and b) an effective amount of one or more NO (nitric oxide) donors.
  • the NO donor is selected from the group consisting of an organic nitrate, a diazeniumdiolate (NONOate), an S-nitrosothiol, an active pharmaceutical agent comprising or having been derivatized with an NO group, an NO-zeolite, arginine, sodium nitroprusside (SNP), and any combination thereof.
  • NONOate diazeniumdiolate
  • SNP sodium nitroprusside
  • At least one of the NO donors is an organic nitrate.
  • the organic nitrate is selected from the group consisting of:
  • ISMN m ononitrate
  • PETN pentaerythritol tetranitrate
  • At least one of the NO donors is a diazeniumdiolate
  • the diazeniumdiolate is selected from the group consisting of
  • At least one of the NO donors is an S-nitrosothiol.
  • the S-nitrosothiol is selected from the group consisting of
  • At least one of the NO donors is a pharmaceutical agent comprising one or more NO groups, such as a pharmaceutical agent modified to be covalently or non-covalently bound to one or more NO groups.
  • at least one of the NO groups is a nitro-oxy group, an S-nitrosothiol group, or a furoxan group.
  • at least one of the NO groups is a nitro-oxy group.
  • at least one of the NO groups is a nitro-oxy group.
  • At least one of the NO groups is an S-nitrosothiol group
  • At least one of the NO groups is a furoxan group, and the NO
  • the pharmaceutical agent comprising one or more NO groups is any pharmaceutical agent that has been modified to comprise one or more NO groups, such as a PDE inhibitor comprising one or more NO groups, a biguanide comprising one or more NO groups, or a sirtuin pathway activator comprising one or more NO groups, or any combination thereof.
  • the PDE inhibitor comprising one or more NO groups is a PDE5 inhibitor comprising one or more NO groups.
  • the PDE5 inhibitor comprising one or more NO groups is sildenafil comprising one or more NO groups.
  • the biguanide comprising one or more NO groups is metformin comprising one or more NO groups.
  • the sirtuin pathway activator comprising one or more NO groups is a PGC- ⁇ activator comprising one or more NO groups.
  • the PGC-la activator comprising one or more NO groups is a thiazolidinedione comprising one or more NO groups.
  • the thiazolidinedione comprising one or more NO groups is rosiglitazone comprising one or more NO groups.
  • At least one of the NO donors is arginine. In other embodiments, at least one of the NO donors is L-arginine. In certain embodiments, at least one of the NO
  • At least one of the NO donors has L J (nitroprusside).
  • at least one of the NO donors is sodium nitroprusside (SNP).
  • the one or more NO donors are present at an amount between about 0.01 to about 1 wt% of the total weight of the composition. In certain embodiments, the one or more NO donors are present at an amount of about 0.05 mg to about 10 g. In certain embodiments, the composition comprises about 100 mg to about 10 g L-arginine. In certain embodiments, the composition comprises about 0.05 mg to about 10 mg sodium nitroprusside (SNP). In certain embodiments, the leucine metabolites is ⁇ -hydroxy ⁇ -methylbutyrate (HMB) and/or keto-isocaproic acid (KIC).
  • HMB ⁇ -hydroxy ⁇ -methylbutyrate
  • KIC keto-isocaproic acid
  • a) leucine and/or a leucine metabolite is present at an amount between about 50 to about 95 wt% of the total weight of the composition. In certain embodiments, the composition comprises at least about 500 mg leucine and/or at least about 200 mg leucine metabolite.
  • the composition further comprises at least a PDE inhibitor, a biguanide, a sirtuin pathway activator, or any combination thereof.
  • the composition further comprises a PDE inhibitor.
  • the PDE inhibitor is a PDE5 inhibitor selected from the group consisting of icariin, sildenafil, tadalafil, vardenafil, avanafil, lodenafil, mirodenafil and udenafil.
  • the composition further comprises a biguanide.
  • the biguanide is metformin.
  • the composition further comprises a sirtuin pathway activator.
  • the sirtuin pathway activator is a PGC-la activator.
  • the PGC-la activator is a thiazolidinedione.
  • the thiazolidinedione is rosiglitazone.
  • the composition further comprises at least about 0.1 mg sildenafil. In certain embodiments, the composition further comprises sildenafil with an amount between about 0.01 to about 1 wt% of the total weight of the composition. In certain
  • the composition further comprises metformin with an amount between about 5 to about 50 wt% of the total weight of the composition. In certain embodiments, the composition further comprises about 1 g to about 2.55 g metformin. In certain embodiments, the composition further comprises about 0.5 g to about 1.25 g metformin. In certain embodiments, the composition further comprises about 20 to about 1000 mg metformin. In certain embodiments, the composition further comprises 10 to 500 mg metformin. In certain embodiments, the composition further comprises about 0.1 to about 4 mg rosiglitazone. In certain embodiments, the composition is formulated as a unit dose comprising about 500 to about 1200 mg of leucine, about 200 to about 1,000 mg of metformin, and about 0.05 mg to about 10 mg of an NO donor.
  • the composition further comprises a pharmaceutically acceptable carrier.
  • the composition is formulated for oral, nasal, transdermal or injectable administration.
  • the composition is a liquid form.
  • the composition is a solid form.
  • the composition is formulated as a tablet, chewable tablet, caplets, capsule, soft gelatin capsules, lozenges, spray, a transdermal patch, or solution.
  • a method of preventing or reducing nonalcoholic steatohepatitis (NASH) in a subject in need thereof comprising administering to the subject any composition described herein.
  • a method of preventing or reducing non-alcoholic steatohepatitis (NASH) in a subject in need thereof comprising: administering to the subject a) a composition comprising leucine and/or a metabolites of leucine; and b) a composition comprising one or more NO (nitric oxide) donors separately or simultaneously.
  • the method further comprises
  • the method further comprises administering to the subject a composition comprising a PDE inhibitor, a composition comprising a biguanide, a composition comprising a sirtuin pathway activator, or any combination thereof.
  • the method further comprises administering to the subject a composition comprising a PDE inhibitor.
  • the method further comprises administering to the subject a composition comprising a biguanide.
  • the method further comprises administering to the subject a composition comprising a sirtuin pathway activator.
  • the PDE inhibitor is a PDE5 inhibitor selected from the group consisting of icariin, sildenafil, tadalafil, vardenafil, avanafil, lodenafil, mirodenafil and udenafil.
  • the biguanide is metformin.
  • the sirtuin pathway activator is a PGC- ⁇ activator.
  • the PGC- ⁇ activator is a thiazolidinedione.
  • the thiazolidinedione is rosiglitazone.
  • any of the compositions disclosed herein further comprise a pharmaceutically acceptable carrier.
  • any of the compositions disclosed herein is formulated for oral, nasal, or injectable administration.
  • any of the compositions disclosed herein is a liquid form.
  • any of the compositions is a solid form.
  • any of the compositions is formulated as a tablet, chewable tablet, caplets, capsule, soft gelatin capsules, lozenges, spray, a transdermal patch, or solution.
  • any of the compositions is administered 1, 2, 3, 4, or 5 per day.
  • any of the methods can comprise administering leucine 1, 2, 3, 4, or 5 times per day.
  • any of the methods can comprise administering at least one of the NO donors 1, 2, 3, 4, or 5 times per day. In certain embodiments, any of the methods can comprise administering metformin 1, 2, 3, 4, or 5 times per day. In certain embodiments, any of the compositions can be administered to the subject transdermally, orally or by inhalation.
  • the subject has been diagnosed with the non-alcoholic steatohepatitis (NASH), or exhibits a non-alcoholic fatty liver disease (NAFLD) selected from the group consisting of non-alcoholic fatty liver (NAFL), NASH-related cirrhosis and hepatic steatosis.
  • NASH non-alcoholic fatty liver
  • the non-alcoholic steatohepatitis (NASH) in the subject is evidenced by an accumulation of hepatic fat, fibrosis and/or inflammation.
  • the accumulation of hepatic fat, fibrosis and/or inflammation is detectable by one or more methods selected from the group consisting of ultrasonography, computed tomography (CT), magnetic resonance imaging, magnetic resonance spectroscopy (MRS), magnetic resonance elastography (MRE), transient elastography (TE), hepatic steatosis, inflammation and/or fibrosis detected by biopsy, and detection of a biomarker indicative of liver damage.
  • the biomarker indicative of liver damage is elevated serum alanine aminotransferase, aspartate aminotransferase, and/or cytokeratin 18.
  • the reduction of non-alcoholic steatohepatitis (NASH) in the subject is evidenced by a reduction in hepatic fat.
  • the reduction in hepatic fat is detectable by one or more methods selected from the group consisting of ultrasonography, computed tomography (CT), magnetic resonance imaging measurement of serum alanine transaminase and aspartate transaminase, and biopsy.
  • CT computed tomography
  • the subject exhibits a non-alcoholic fatty liver disease (NAFLD).
  • the NAFLD is selected from the group consisting of non-alcoholic fatty liver (NAFL), NASH-related cirrhosis and hepatic steatosis.
  • the reduction of non-alcoholic steatohepatitis (NASH) in the subject is characterized by a reduction in hepatic steatosis, inflammation, ballooning, and/or fibrosis.
  • the method further comprises determining the subject as having nonalcoholic steatohepatitis (NASH) before administering to the subject a composition comprising leucine and/or a metabolite of leucine.
  • the method further comprises determining the subject as having non-alcoholic steatohepatitis (NASH) before administering to the subject a composition comprising at least one of the NO donors.
  • Subjects benefit from any of the foregoing inventions can include, but are not limited to, subjects who had been diagnosed with or exhibit a propensity to non-alcoholic steatohepatitis (NASH), and subjects who had been diagnosed or exhibit a non-alcoholic fatty liver disease (NAFLD), such as non-alcoholic fatty liver, non-alcoholic steatohepatitis (NASH), and NASH- related cirrhosis.
  • NASH-related cirrhosis In some cases, the subject has non-alcoholic steatohepatitis (NASH).
  • the pharmaceutical composition for administration in any of the foregoing methods is substantially free of one or more free amino acids selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.
  • free amino acids selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.
  • kits comprising a multi-day supply of unit dosages of a composition described herein and instructions directing the administration of the multi-day supply over a period of multiple days.
  • the kit further comprises a wearable activity monitor for administration.
  • the multi-day supply can be a two-day, three-day, five-day, six-day, seven-day, two-week, one-month, two-month, 90-day, 180-day, 3-month or 6- month supply.
  • kits comprising a composition comprising: a) leucine and/or a metabolite of leucine; and b) one or more NO (nitric oxide) donors.
  • a kit comprising any of the compositions described herein.
  • any of the compositions can be separated packaged, for example, a) leucine and/or a metabolite of leucine; and b) one or more NO (nitric oxide) donors are separately packaged; or two or more of the compositions described herein can be packaged together in a container.
  • kits for of preventing or reducing non-alcoholic steatohepatitis comprising: a) a container comprising a composition comprising leucine and/or a metabolite of leucine; and b) a separate container comprising a composition comprising one or more NO (nitric oxide) donors, and optionally one or more additional containers comprising one or more of the compositions described herein, such as a composition comprising at least a PDE inhibitor, a composition comprising at least a biguanide, a composition comprising a sirtuin pathway activator, or any combination thereof.
  • NASH non-alcoholic steatohepatitis
  • the NO donors can be any of the NO donors described herein or any suitable NO donors, such as L-arginine or SNP.
  • the leucine metabolite is ⁇ -hydroxy ⁇ -methylbutyrate (HMB) and/or keto-isocaproic acid (KIC).
  • the PDE inhibitor is a PDE5 inhibitor selected from the group consisting of icariin, sildenafil, tadalafil, vardenafil, avanafil, lodenafil, mirodenafil and udenafil.
  • the biguanide is metformin.
  • the sirtuin pathway activator is a PGC- ⁇ activator.
  • the PGC- ⁇ activator is a thiazolidinedione.
  • the thiazolidinedione is rosiglitazone.
  • Figure 1 depicts a graph showing the synergistic effects of sodium nitroprusside (SNP; 10 ⁇ ) and leucine (Leu; 0.5 mM) on nitric oxide production in human vascular endothelial cells.
  • SNP sodium nitroprusside
  • Leu leucine
  • Figure 2 depicts a graph showing the synergistic effects of arginine (Arg; 1 ⁇ ) and leucine (Leu; 0.5 mM) on nitric oxide production on human vascular endothelial cells.
  • Figure 3 depicts a graph showing the synergistic effects of sodium nitroprusside (SNP; 10 ⁇ ) and leucine (Leu; 0.5 mM) on reducing hepatocyte lipids.
  • SNP sodium nitroprusside
  • Leu leucine
  • Figure 4 depicts a graph showing the synergistic effects of arginine (Arg; 1 ⁇ ) and leucine (Leu; 0.5 mM) on reducing hepatocyte lipids.
  • nitric oxide donor or “NO donor” refers to compounds suitable to increase nitric oxide (NO) levels or bioactivity directly or indirectly, e.g., by increasing the amount or level of available substrate or enzymes for NO production.
  • nitric oxide donors include compounds that release NO or a related redox species under physiological or any disease treatment conditions, compounds that provide nitric oxide bioactivity, e.g., vasorelaxation or stimulation or inhibition of a receptor protein, or compounds, after non-enzymatic or enzymatic reaction, release nitric oxide.
  • a "NO donor” may or may not include a nitric oxide chemical group.
  • L- arginine particularly examples thereof include L- arginine, SNP, organic nitrate, compounds inhibiting arginase, e.g. arginase II, a competitor of the NO synthase (NOS).
  • Other examples comprise compounds stimulating the activity of NO synthase, in particular, of the eNOS, either by increasing the amount or level of NOS or the turnover rate of the enzyme. These compounds may act directly or indirectly through affecting the level of natural eNOS stimulators, like sphinogine 1-phospate.
  • organic nitric oxide donor means an organic compound or mixture of compounds with at least one of such compound(s) which are NO donors, such as compound that can release nitric oxide under physiological or any disease treatment conditions.
  • an "organic nitrate” refers to a compound of formula R-ON0 2 , wherein R is an alkyl, alkenyl, alkynyl, aryl, arylalkyl, arylalkenyl, arylalkynyl, alkylaryl, alkenylaryl or alkynylaryl group, each of which can optionally be substituted, optionally with one or more further -ON0 2 groups, and each of which may optionally include one or more heteroatoms N, O or S in its carbon skeleton.
  • an "organic nitrite” refers to a compound of formula R b -ONO, wherein R b is an alkyl, alkenyl, alkynyl, aryl, arylalkyl, arylalkenyl, arylalkynyl, alkylaryl, alkenylaryl or alkynylaryl group, each of which may optionally be substituted, optionally with one or more further -ONO groups, and each of which may optionally include one or more heteroatoms N, O or S in its carbon skeleton.
  • a cell includes a plurality of cells, including mixtures thereof.
  • interrogating or “analyzing” are used interchangeably herein to refer to any form of measurement, and include determining if an element is present or not. These terms include both quantitative and/or qualitative determinations that can be relative or absolute.
  • the term "subject” or “individual” includes mammals. Non-limiting examples of mammals include humans and mice, including transgenic and non-transgenic mice. The methods described herein can be useful in both human therapeutics, pre-clinical, and veterinary applications. In some embodiments, the subject is a mammal, and in some
  • the subject is human.
  • Other mammals include, and are not limited to, apes, chimpanzees, orangutans, monkeys; domesticated animals (pets) such as dogs, cats, guinea pigs, hamsters, mice, rats, rabbits, and ferrets; domesticated farm animals such as cows, buffalo, bison, horses, donkey, swine, sheep, and goats; or exotic animals typically found in zoos, such as bear, lions, tigers, panthers, elephants, hippopotamus, rhinoceros, giraffes, antelopes, sloth, gazelles, zebras, wildebeests, prairie dogs, koala bears, kangaroo, pandas, giant pandas, hyena, seals, sea lions, and elephant seals.
  • domesticated animals such as dogs, cats, guinea pigs, hamsters, mice, rats, rabbits, and ferret
  • agent refers to a biological, pharmaceutical, or chemical compound or other moiety.
  • Non-limiting examples include simple or complex organic or inorganic molecule, a peptide, a protein, a peptide nucleic acid (PNA), an oligonucleotide (including e.g., aptomer and polynucleotides), an antibody, an antibody derivative, antibody fragment, a vitamin derivative, a carbohydrate, a toxin, a branched chain amino acid in free amino acid form or metabolite thereof, or a chemotherapeutic compound.
  • PNA peptide nucleic acid
  • oligonucleotide including e.g., aptomer and polynucleotides
  • oligomers e.g., oligopeptides and oligonucleotides
  • synthetic organic compounds based on various core structures.
  • natural sources can provide compounds for screening, such as plant or animal extracts, and the like. A skilled artisan can readily recognize that there is no limit as to the structural nature of the agents.
  • administer means administering a composition to a subject via a route known in the art, including but not limited to intravenous, intraarterial, oral, parenteral, buccal, topical, transdermal, rectal, intramuscular, subcutaneous, intraosseous, transmucosal, or intraperitoneal routes of
  • oral routes of administering a composition can be used.
  • co-administration can encompass administration of two or more agents to an animal so that both agents and/or their metabolites are present in the subject at the same time.
  • Co-administration can encompass treatment regimens in which the composition and additional therapeutic agent are administered by the same or different route of administration or at the same or different times.
  • Co-administration includes simultaneous administration in separate compositions, administration at different times in separate
  • compositions or administration in a composition in which both agents are present.
  • Coadministration can include simultaneous administration of the agents in separate compositions, administration at different times in separate compositions, and/or administration in a single composition comprising each of the agents to be co-administered.
  • the term "effective amount” or “therapeutically effective amount” refers to that amount of an agent described herein that is sufficient to effect the intended application including but not limited to disease treatment, as defined below.
  • the therapeutically effective amount can vary depending upon the intended application ⁇ in vitro or in vivo), or the subject and disease condition being treated, e.g., the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art.
  • the term can also apply to a dose that can induce a particular response in target cells, e.g., reduction of proliferation or down regulation of activity of a target protein.
  • the specific dose can vary depending on the particular compounds chosen, the dosing regimen to be followed, whether it is administered in combination with other compounds, timing of administration, the tissue to which it is administered, and the physical delivery system in which it is carried.
  • energy metabolism refers to the transformation of energy that accompanies biochemical reactions in the body, including cellular metabolism and mitochondrial biogenesis. Energy metabolism can be quantified using the various measurements described herein, for example and without limitations, weight-loss, fat-loss, insulin sensitivity, fatty acid oxidation, glucose utilization, triglyceride content, Sirt 1 expression level, AMPK expression level, oxidative stress, and mitochondrial biomass.
  • isolated refers to a preparation of the substance devoid of at least some of the other components that may also be present where the substance or a similar substance naturally occurs or is initially obtained from.
  • an isolated substance may be prepared by using a purification technique to enrich it from a source mixture.
  • Enrichment can be measured on an absolute basis, such as weight per volume of solution, or it can be measured in relation to a second, potentially interfering substance present in the source mixture. In certain embodiments, increasing enrichment of any of the components can be used. Thus, for example, a 2-fold enrichment, 10-fold enrichment, 100-fold enrichment, or 1000-fold enrichment can be used.
  • a substance can also be provided in an isolated state by a process of artificial assembly, such as by chemical synthesis.
  • a "modulator" of a pathway refers to a substance or agent which modulates the activity of one or more cellular proteins mapped to the same specific signal transduction pathway.
  • a modulator can augment or suppress the activity and/or expression level or pattern of a signaling molecule.
  • a modulator can activate a component in a pathway by directly binding to the component.
  • a modulator can also indirectly activate a component in a pathway by interacting with one or more associated components.
  • the output of the pathway can be measured in terms of the expression or activity level of proteins.
  • the expression level of a protein in a pathway can be reflected by levels of corresponding mRNA or related transcription factors as well as the level of the protein in a subcellular location.
  • certain proteins can be activated by translocating in or out of a specific subcellular component, including but not limited to nucleus, mitochondria, endosome, lysosome or other membranous structure of a cell.
  • the output of the pathway can also be measured in terms of physiological effects, such as mitochondrial biogenesis, fatty acid oxidation, or glucose uptake.
  • a “pathway activator” refers to a modulator that influences a pathway in a manner that increases the pathway output. Activation of a particular target may be direct (e.g. by interaction with the target) or indirect (e.g. by interaction with a protein upstream of the target in a signaling pathway including the target).
  • compositions that have less than about 10%, less than about 5%, less than about 1%, less than about 0.5%, less than 0.1% or even less of a specified component refers to compositions that have less than about 1% of the non-branched chain amino acid lysine.
  • substantially free of a non-branched chain amino acid can be evidenced by less than 1% of the non-branched chain amino acid when compared to the rest of the amino acids in a given composition.
  • a "sub-therapeutic amount" of an agent, an NO donor, an activator, an inhibitor or a therapy is an amount less than the effective amount for that agent, NO donor, activator, inhibitor, or therapy, but when combined with an effective or sub -therapeutic amount of another agent, NO donor, activator, inhibitor, or therapy can produce a desired result, due to, for example, synergy in the resulting efficacious effects, and/or reduced side effects.
  • a sub-therapeutic amount of the agent or component can be such that it is an amount below which would be considered therapeutic. For example, FDA guidelines can suggest a specified level of dosing to treat a particular condition, and a sub-therapeutic amount would be any level that is below the FDA suggested dosing level.
  • the sub-therapeutic amount can be about 1, 5, 10, 15, 20, 25, 30, 35, 50, 75, 90, or 95% less than the amount that is considered to be a therapeutic amount.
  • the therapeutic amount can be assessed for individual subjects, or for groups of subjects.
  • the group of subjects can be all potential subjects, or subjects having a particular characteristic such as age, weight, race, gender, or physical activity level.
  • a "synergistic" or “synergizing” effect can be such that the one or more effects of the combination compositions are greater than the one or more effects of each component alone at a comparable dosing level, or they can be greater than the predicted sum of the effects of all of the components at a comparable dosing level, assuming that each component acts independently.
  • the synergistic effect can be about, or greater than about 10, 20, 30, 50, 75, 100, 110, 120, 150, 200, 250, 350, or 500% or even more than the effect on a subject with one of the components alone, or the additive effects as measured when each of the components when administered individually.
  • the effect can be any of the measurable effects described herein.
  • free amino acid can refer to amino acids that are not bound to other amino acids, for example, by peptide bonds.
  • free or “individual” leucine refers to leucine not bound to other amino acids by peptide bonds.
  • a "biological marker” or a “biomarker” generally refers to a measurable indicator of some biological state or condition. Biological markers are often measured and evaluated to examine normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention. Accordingly, the bioniarkers can relate to genes, mRNAs, and proteins corresponding to the biomarkers as described herein.
  • the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.
  • A, B, C, or combinations thereof refers to all permutations and combinations of the listed items preceding the term.
  • A, B, C, or combinations thereof is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.
  • expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, MB, BBC, AAABCCCC, CBBAAA, CAB ABB, and so forth.
  • the skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.
  • compositions and methods have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
  • Certain embodiments provide a) a branched chain amino acid; and b) one or more NO (nitric oxide) donors.
  • the NO donor is selected from the group consisting of an organic nitrate, a diazeniumdiolate (NONOate), an S-nitrosothiol, an active pharmaceutical agent that has been derivatized with an NO group, an NO-zeolite, arginine, sodium nitroprusside (SNP), and any combination thereof
  • the branched chain amino acid and/or metabolite thereof is leucine.
  • the composition comprises an additional agent.
  • the additional agent is one or more sirtuin pathway activators.
  • the additional agent is metformin, and/or a PDE inhibitor, e.g. sildenafil.
  • co-administration of the NO donor and the branched chain amino acid and/or metabolite thereof and, optionally, the additional agent reduces or prevents NASH or hepatic steatosis to a greater extent than administration of any one of the agents alone.
  • co-administration of the NO donor and branched chain amino acid in free amino acid form (or metabolite thereof) and, optionally, the additional agent has a synergistic effect, e.g., reduces or prevents NASH or hepatic steatosis to a greater extent than an additive effect of administering the NO donor alone, administering the branched chain amino acid in free amino acid form (or metabolite thereof) alone and administering the additional agent alone.
  • the composition is formulated in tablet or capsules for administration to a subject in need thereof.
  • the subject may have NASH or hepatic steatosis.
  • the subject is diagnosed with NASH or hepatic steatosis, NAFLD or NASH-related cirrhosis.
  • the subject exhibits a propensity for having NASH or hepatic steatosis, NAFLD or NASH-related cirrhosis.
  • the composition can be formulated for oral administration.
  • the composition can comprise at least one tablet or capsule.
  • the composition can comprise at least two tablets or capsules.
  • the composition comprises three tablets or capsules, four tablets or capsules, five tablets or capsules, or more.
  • the composition is administered at least once a day, twice a day, three times a day, or more.
  • the tablets or capsules in a composition can comprise the same amount or different amount of each of the component.
  • the composition can comprise the same amount or different amount of leucine in the form of a free amino acid or metabolite thereof.
  • the composition can comprise the same amount or different amount of a sirtuin pathway activator such as metformin.
  • the composition can comprise the same amount or different amount of a PDE5 inhibitor such as sildenafil.
  • a tablet or capsule of the composition described herein comprises leucine in the form of a free amino acid and/or leucine metabolite and an NO donor. In some embodiments, a tablet or capsule of the composition comprises leucine and/or leucine metabolite thereof and metformin. In some, a tablet or capsule of the composition comprises leucine in the form of a free amino acid or metabolite thereof, metformin and an NO donor. In some embodiments, a tablet or capsule of the composition comprises leucine in the form of a free amino acid or leucine metabolite, an NO donor, metformin and sildenafil.
  • a composition described herein comprises (a) an amount of leucine in the form of a free amino acid or metabolite thereof, and (b) an amount of a NO donor. In certain embodiments, the amount of in the form of a free amino acid or metabolite thereof administered in a tablet or capsule that does not contain an NO donor. In some embodiments, the composition further comprises a third component, (c) an amount of an additional active pharmaceutical ingredient, which may be metformin, sildenafil, any other active pharmaceutical ingredient, or any combination thereof.
  • the amount of leucine or a leucine metabolite, excluding fillers is between about 50-95 wt% of a total wt of (a), (b) and (c) or the composition. In certain embodiments, the amount of leucine or a leucine metabolite, excluding fillers, is between about 0.1-1% of a total wt of (a), (b) and (c) or the total
  • the amount of leucine or a leucine metabolite, excluding fillers is greater than or about 0.01, 0.1, 0.5, 1, 2, or 5 % of a total wt of (a), (b) and (c) or the total composition. In certain embodiments, the amount of leucine or a leucine metabolite, excluding fillers, is 5-50 wt% of a total wt of (a), (b), and (c) or the total composition. In certain embodiments, the amount of an NO donor, excluding fillers, is between about 0.1-1% of a total wt of (a), (b) and (c) or the total composition.
  • the amount of an NO donor, excluding fillers is between about 1-5% of a total wt of (a), (b) and (c) or the total composition. In certain embodiments, the amount of an NO donor, excluding fillers, is less than or about 0.01, 0.1, 0.5, 1, 2, or 5 % of a total wt of (a), (b) and (c) or the total composition. In certain embodiments, the amount of an NO donor, excluding fillers, is 5-50 wt% of a total wt of (a), (b), and (c) or the total composition.
  • the amount of the additional active pharmaceutical ingredient which may be sildenafil and/or metformin, excluding fillers, is between about 5-50 wt% of a total wt of (a), (b), and (c) or the total composition. In certain embodiments, the amount of additional active pharmaceutical ingredient, excluding fillers, is between about 0.01-1 wt% of a total wt of (a), (b), and (c) or the total composition. In certain embodiments, a wt% of the leucine or leucine metabolite in the pharmaceutical composition, excluding fillers, is about 50-95 wt%, or any intermediate ranges or values. In certain
  • a wt% of the NO donors in the pharmaceutical composition, excluding fillers is about 0.01-99 wt%, or any intermediate ranges or values. In certain embodiments, a wt% of the additional active pharmaceutical ingredient in the pharmaceutical composition, excluding fillers, is about 5-50 wt%, or any intermediate ranges or values. In certain embodiments, a wt% of the additional active pharmaceutical ingredient in the pharmaceutical composition, excluding fillers, is about 0.01-1 wt%, or any intermediate ranges or values.
  • compounds for the reduction, the sustained reduction of, and/or prevention of NAFLD, NASH and/or hepatic steatosis are provided.
  • coadministration of a branched chain amino acid in free amino acid form or metabolite thereof, and an additional agent can reduce and/or prevent NAFLD, NASH and/or hepatic steatosis in a subject.
  • the additional agent can be a sirtuin pathway activator and/or a PDE inhibitor, such as a PDE5 inhibitor.
  • the NO donor can be an organic nitrate or nitrite selected from the group consisting of amyl nitrate, ethyl nitrite, ethyl nitrate, isosorbide mononitrate, isosorbide dinitrate,
  • At least one of the NO donors is arginine. In other embodiments, at least one of the NO donors is L-arginine. In certain embodiments,
  • At least one of the NO donors has L J (nitroprusside). In further embodiments, at least one of the NO donors is sodium nitroprusside (SNP).
  • nitric oxide donors for use in certain aspects include without limitation, isosorbide dinitrate, L-arginine, linsidomine, minoxidil, nicorandil, nitroglycerin, nitroprusside, nitrosoglulthathione, and S-nitroso-N-acetyl-penicillamine (SNAP) or glyceryl trinitrate.
  • isosorbide mononitrate, glyceryl trinitrate, or L-arginine is used as organic NO donor.
  • the nitric oxide donor is a compound selected from the group consisting of an organic nitrite, an organic nitrate, a nitrite ester of a polyol, a nitrate ester of a polyol molsidomine and its metabolites, a diazeniumdiolate, a S-nitrosothiol, an iron-sulphur nitrosyl, sodium nitrite, ethylene glycol dinitrate, isopropyl nitrate, amyl nitrite, amyl nitrate, ethyl nitrite, butyl nitrite, isobutyl nitrite, octyl nitrite, glyceryl- 1 -monoitrate, glyceryl-1,2- dinitrate, glyceryl-l,3-dinitrate, nitroglycerin, butane- 1,2,4-triol -tri
  • NO donors include L-arginine.
  • L-arginine is converted in the body into a chemical called nitric oxide.
  • NO donors also include agents that stimulate endogenous NO or elevate levels of endogenous endothelium-derived relaxing factor (EDRF) in vivo or are substrates for nitric oxide synthase.
  • EDRF endogenous endothelium-derived relaxing factor
  • Such compounds include, for example, L- arginine, L-homoarginine, and N-hydroxy-L-arginine, including their nitrosated and nitrosylated analogs (e.g., nitrosated L-arginine, nitrosylated L-arginine, nitrosated N-hydroxy-L-arginine, nitrosylated N-hydroxy-L-arginine, nitrosated L-homoarginine and nitrosylated L- homoarginine), precursors of L-arginine and/or physiologically acceptable salts thereof, including, for example, citrulline, ornithine or glutamine, inhibitors of the enzyme arginase (e.g., N-hydroxy-L-arginine and 2(S)-amino-6-boronohexanoic acid) and the substrates for nitric oxide synthase, cytokines, adenosin, bradykinin, calreticulin, bisac
  • NO donors can be molsidomine and its active metabolite linsidomine, as well as sodium nitroprusside. These substances do not need to be reduced to donate NO.
  • Sodium nitroprusside is an inorganic compound with the formula Na 2 [Fe(CN) 5 NO], usually encountered as the dihydrate, Na 2 [Fe(CN) 5 NO] 2H 2 0. This red-colored sodium salt dissolves in water ethanol to give solutions containing the free complex dianion [Fe(CN) 5 NO] 2 ⁇ .
  • This compound is used as a drug. In this role it is abbreviated SNP, and it has tradenames like Nitropress. It acts as a drug by releasing nitric oxide; it belongs to the class of NO-releasing drugs as a result. This drug is used as a vasodilator to reduce blood pressure.
  • At least one of the NO donors is an organic nitrate.
  • the organic nitrate is selected from the group consisting of: (glyceryl trinitrate (GTN)), NO 2 (isosorbide
  • ISMN mononitrate
  • PETN erythritol tetranitrate
  • the organic nitrates are the most commonly used NO donor drugs.
  • the NO donor is an organic nitrate such as glyceryl trinitrate (GTN; also known as nitroglycerin).
  • GTN is the best-studied nitrate, used mainly in acute relief of pain associated with angina.
  • GTN ointments are also routinely used for the treatment of anal fissure, transdermal patches in heart failure and chronic angina, whereas nebulized GTN may have benefits in certain subgroups with pulmonary hypertension.
  • GTN contains three nitro-oxy ester (referred to as nitrate) groups, but releases only one molar equivalent of NO from the terminal position after bioactivation.
  • the NO donor is an organic nitrate such as isosorbide mononitrate (ISMN) used for the treatment of chronic angina.
  • ISMN isosorbide mononitrate
  • organic nitrates can include BiDil (isosorbide dinitrate with hydralazine).
  • the NO donor includes at least one organic nitrate which includes esters of nitric acid and can be an acyclic or cyclic compound, such as represented by the following general formula: R(-CR'R"-0-N0 2 ) x , wherein: R is an organic or H (hydro) moiety or covalent bond, particularly a 2 to about 12 carbon hydrocarbon or oxygen- substituted hydrocarbon, especially one having 2 to 6 carbons and from 0 to 2 oxygen(s); R is an organic or hydro moiety or covalent bond, and preferably methyl; lower alkyl, to include ethyl, propyl, butyl, pentyl, and hexyl; methoxy; lower alkoxy; or hydro; R" is an organic or hydro moiety or covalent bond, preferably methyl, lower alkyl, methoxy, lower alkoxy, or hydro, and especially hydro; and X is an integer from 1 to about 12, and preferably from 2 to 6.
  • R is an organic or H (hydro) mo
  • the organic nitrate can be ethylene glycol dinitrate, isopropyl nitrate; glyceryl- 1 -mononitrate; glyceiyl-1 ,2-dinitrate; glyceryl-l,3-dinitrate; nitroglycerin (GTN); butane- 1,2,4-triol -trinitrate; erythrityl tetranitrate (ETN); pentaerythrityl tetranitrate (PETN); isosorbide mononitrate (ISMN), which may include isosorbide-2-monotritate (IS2N) and/or isosorbide-5-mononitrate (IS5N); and/or isosorbide dininitrate (ISDN), and so forth and the like.
  • GTN nitroglycerin
  • ISMN isosorbide mononitrate
  • ISMN isosorbide mononitrate
  • ISMN isosorbide mononitrate
  • At least one of the NO donors is a diazeniumdiolate
  • the diazeniumdiolate is selected from the group consisting of
  • any suitable diazeniumdiolates can be used in certain embodiments.
  • the first of this class to be described was an adduct of diethylamine and NO (diethylamine NONOate; DEA/NO).
  • NONOates decompose spontaneously in solution at physiological pH and temperature, to generate up to 2 molar equivalents of NO. The rate of decomposition is dependent on the structure of the nucleophile.
  • a range of NONOates has now been described with half-lives varying from seconds to hours.
  • An attractive feature of this class of compounds is that their decomposition is not catalysed by thiols or biological tissue, unless specifically designed to (see below) and, because NO release follows simple first-order kinetics, the rate of NO release can be accurately predicted.
  • At least one of the NO donors is an S-nitrosothiol.
  • the S-nitrosothiol is selected from the group consisting of:
  • S-nitrosothiol is a class of NO donors which contain a single chemical bond between a thiol (sulphydryl) group (R-SH) and the NO moiety.
  • Biological activity of S-nitrosothiols is highly influenced by the molecular environment of the parent thiol. That said, the complex chemistry of NO release from even the most basic S-nitrosothiol gives these compounds several means by which they can confer NO bioactivity.
  • S-nitrosothiols are considered to be NO + donors and transfer of NO + across the plasma membrane via protein disulphide isomerases may allow even large molecule weight S-nitrosothiols to transfer oxides of nitrogen across cell membranes to subcellular targets.
  • S-nitrosothiols have advantages over other classes of NO donors, such as the nitrates, as they have far less stringent metabolic requirements and this may be the reason that they do not induce tolerance with long-term use.
  • S-nitrosothiols have a number of potential advantages over other classes of NO donors. Firstly, some examples show tissue selectivity: S-nitroso-glutathione (GSNO) is selective for arteries over veins, giving them a different haemodynamic profile of action than those of classical organic nitrates. Additionally, S-nitrosothiols are potent antiplatelet agents, inhibiting aggregation at doses that do not influence vascular tone. Furthermore, the ability of S- nitrosothiols to directly transfer NO species allows biological activity to be passed on through a chain of other thiols without the release of free NO. This mechanism of bioactivation may make S-nitrosothiols less susceptible to conditions of oxidative stress by effectively protecting the NO moiety from attack by oxygen-centered free radicals.
  • the nitric oxide donor is a small molecule.
  • O-nitroso compounds are compounds having one or more -O-NO groups, and are also referred to as O-nitrosylated compounds and nitrite compounds.
  • S-nitroso compounds are compounds with one or more -S-NO groups and are also referred to as nitrosothiols and S-nitrosylated compounds.
  • An -S-NO group is also referred to in the art as a sulfonyl nitrite, a thionitrous acid ester, an S-nitrosothial or a thionitrite.
  • NO compound includes NONOates, nitroprusside, nitrates, furoxans, etc.
  • nitro compounds -Y-N0 2 are included in the embodiment (where Y is N,
  • At least one of the NO donors is a pharmaceutical agent comprising one or more NO groups, such as a pharmaceutical agent modified to be covalently or non-covalently bound to one or more NO groups.
  • at least one of the NO groups is a nitro-oxy group, an S-nitrosothiol group, or a furoxan group.
  • at least one of the NO groups is a nitro-oxy group.
  • at least one of the NO groups is a nitro-oxy group.
  • At least one of the NO groups is an S-nitrosothiol group
  • NO donor can include (SNO-diclofenac) and/or
  • At least one of the NO groups is a furoxan group, and the NO
  • donor can (furoxan bound to 4-pheny 1-1, 4-dihydropyri dine).
  • the pharmaceutical agent comprising one or more NO groups is a PDE inhibitor comprising one or more NO groups, a biguanide comprising one or more NO groups, or a sirtuin pathway activator comprising one or more NO groups, or any combination thereof.
  • the PDE inhibitor comprising one or more NO groups is a PDE5 inhibitor comprising one or more NO groups.
  • the PDE5 inhibitor comprising one or more NO groups is sildenafil comprising one or more NO groups.
  • the biguanide comprising one or more NO groups is metformin comprising one or more NO groups.
  • the sirtuin pathway activator comprising one or more NO groups is a PGC- ⁇ activator comprising one or more NO groups.
  • the PGC-la activator comprising one or more NO groups is a thiazolidinedione comprising one or more NO groups.
  • the thiazolidinedione comprising one or more NO groups is rosiglitazone comprising one or more NO groups.
  • One or more branched chain amino acids may be used with one or more NO donors in compositions or methods for co-administration.
  • Branched chain amino acids may have aliphatic side chains with a branch carbon atom that is bound to two or more other atoms. The other atoms may be carbon atoms.
  • Examples of branched chain amino acids include leucine, isoleucine, and valine.
  • Branched chain amino acids may also include other compounds, such as 4- hydroxyisoleucine.
  • Such branched chain amino acids may be administered to a subject in free amino acid form.
  • the branched chain amino acid in free amino acid form is leucine in free amino acid form.
  • a composition comprising a branched chain amino acid in free amino acid form is substantially free of one or more, or all of non- branched chain amino acids.
  • the compositions are substantially free of one or more, or all of non-branched chain amino acids in free amino acid form.
  • the composition can be substantially free of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, and/or tyrosine.
  • the composition can be substantially free of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, and/or tyrosine in free amino acid form.
  • the composition is substantially free of isoleucine and/or valine in free form.
  • a method described herein can include administration of a salt, derivative, metabolite, catabolite, anabolite, precursor, and/or analog of any branched chain amino acids.
  • the metabolite can be a metabolite of leucine, such as HMB.
  • Metabolites of branched chain amino acids can include hydroxymethylbutyrate (HMB), oc-hydroxyisocaproic acid, and keto-isocaproic acid (KIC), keto isovalerate, and keto isocaproate.
  • Non-limiting exemplary anabolites of branched chain amino acids can include glutamate, glutamine, threonine, oc-ketobytyrate, oc-aceto-oc-hydroxy butyrate, a, -dihydroxy- -methylvalerate, oc- keto-P-methylvalerate, , ⁇ -dihydroxy isovalerate, and oc-keto isovalerate.
  • any of the compositions to be administered to a subject can be formulated such that they do not contain (or exclude) one or more amino acids selected from the group consisting of lysine, glutamate, proline, arginine, valine, isoleucine, aspartic acid, asparagine, glycine, threonine, serine, phenylalanine, tyrosine, histidine, alanine, tryptophan, methionine, glutamine, taurine, carnitine, cystine and cysteine.
  • one or more amino acids selected from the group consisting of lysine, glutamate, proline, arginine, valine, isoleucine, aspartic acid, asparagine, glycine, threonine, serine, phenylalanine, tyrosine, histidine, alanine, tryptophan, methionine, glutamine, taurine, carnitine, cystine and cysteine.
  • compositions disclosed herein can be formulated such that they do not contain (or exclude) one or more free amino acids selected from the group consisting of lysine, glutamate, proline, arginine, valine, isoleucine, aspartic acid, asparagine, glycine, threonine, serine, phenylalanine, tyrosine, histidine, alanine, tryptophan, methionine, glutamine, taurine, carnitine, cystine and cysteine.
  • a composition does not contain any non-branched chain amino acids.
  • a composition does not contain any non-branched chain amino acids in free amino acid form.
  • the mass or molar amount of a non-branched chain amino acid in a composition can be less than 0.01, 0.1, 0.5, 1, 2, or 5 % of the total composition.
  • the mass or molar amount of a non-branched chain amino acid in free amino acid form can be less than 0.01, 0.1, 0.5, 1, 2, or 5 % of the total composition.
  • the mass or molar amount of any branched-chain amino acid or metabolite thereof, aside from leucine or its metabolites can be less than 0.01, 0.1, 0.5, 1, 2, or 5 % of the total composition.
  • the mass or molar amount of any branched-chain amino acid in free amino acid form or metabolite thereof, aside from leucine or its metabolites can be less than 0.01, 0.1, 0.5, 1, 2, or 5 % of the total composition.
  • compositions or methods described herein particularly the compositions including one or more NO donors and one or more branched amino acids such as leucine and/or a leucine metabolite.
  • Sirtuins are highly conserved protein deacetylases and/or ADP-ribosyltransferases that have been shown to extend lifespan in lower model organisms, such as yeast, C. elegans, and drosophila. In mammals, sirtuins have been shown to act as metabolic sensors, responding to environmental signals to coordinate the activity of genes that regulate multiple energy homeostasis pathways. For example, studies have shown that sirtuin activation mimics the effects of caloric restriction, an intervention demonstrated to significantly extend lifespan, and activates genes that improve glucose homeostasis and the conversion of fat to energy by fatty acid oxidation.
  • the sirtuin pathway can be defined as any pathway incorporating or converging upon pathways mediated by phosphodiesterases (PDEs).
  • PDEs are enzymes that interact with cyclic adenosine monophosphates (cAMPs) and cyclic guanosine monophosphates (cGMPs).
  • cAMPs cyclic adenosine monophosphates
  • cGMPs cyclic guanosine monophosphates
  • the PDE family of enzymes comprises multiple subclasses, including PDE 1-11 in humans. Inhibitors of these phosphodiesterases can prevent the inactivation of cAMPs and cGMPs, and can have a variety of different physiological effects.
  • the PDE inhibitors can be selective, by preferentially inhibiting one PDE subclass as compared to another subclass, or no n- selective, which have a substantially lower degree of selectivity for individual PDE subclasses.
  • Sildenafil is an example of a selective PDE inhibitor that has shown selective inhibition of PDE 5.
  • Sildenafil is a pharmaceutically active agent that has been used to treat pulmonary hypertension, erectile dysfunction, and altitude sickness.
  • Sirtuin pathway activators can include any agents which activate one or more components of a sirtuin pathway.
  • the sirtuin pathway includes, without limitation, signaling molecules such as, Sirtl, Sirt3, and AMPK.
  • the output of the pathway can be determined by the expression level and/or the activity of the pathway and/or a physiological effect.
  • activation of the Sirtl pathway includes stimulation of PGCl-a and/or subsequent stimulation of mitochondrial biogenesis and fatty acid oxidation.
  • An increase or activation of a sirtuin pathway can be observed by an increase in the activity of a pathway component protein.
  • the protein can be Sirtl, PGCl-a, AMPK, Epacl, Adenylyl cyclase, Sirt3, or any other proteins and their respective associated proteins along the signaling pathway depicted in FIG. 1 of Park et al., Resveratrol Ameliorates Aging-Related Metabolic Phenotypes by Inhibiting cAMP Phosphodiesterases, Cell 148, 421-433 February 3, 2012.
  • Non-limiting examples of physiological effects that can serve as measures of sirtuin pathway output include mitochondrial biogenesis, fatty acid oxidation, glucose uptake, palmitate uptake, oxygen consumption, carbon dioxide production, weight loss, heat production, visceral adipose tissue loss, respiratory exchange ratio, insulin sensitivity, inflammation marker level, vasodilation, browning of fat cells, and irisin production.
  • Examples of indicia of browning of fat cells include, without limitation, increased fatty acid oxidation, and expression of one or more brown-fat-selective genes (e.g., Ucpl, Cidea, Prdml6, and Ndufsl).
  • changes in one or more physiological effects that can serve as measures of sirtuin pathway output are induced by increasing irisin production.
  • An increase in mitochondrial biogenesis can be evidenced by an increase in the formation of new mitochondria and/or by an increase in mitochondrial functions, such as increased fatty acid oxidation, increased heat generation, increased insulin sensitivity, increased glucose uptake, increased vasodilation, decreased weight, decreased adipose volume, and decreased inflammatory response or biomarkers in a subject.
  • the sirtuin pathway activator is a sirtuin activator.
  • the sirtuin activator can be a Sirtl activator, a Sirt2 activator, and/or Sirt3 activator.
  • Sirtl activity can be determined by measuring deacetylation of a substrate, which can be detected using a
  • the substrate for measuring SIRT1 activity can be any substrate known in the art (for example a peptide containing amino acids 379-382 of human p53 (Arg-His-Lys-Lys[Ac]).
  • the substrate for measuring SIRT3 activity can be any substrate known in the art (for example a peptide containing amino acids 317-320 of human p53 (Gin- Pro— Lys- Lys[Ac])).
  • Exemplary sirtuin activators can include those described in Howitz et al. (2003) Nature 425: 191 and include, for example, resveratrol (3,5,4'-Trihydroxy-trans-stilbene), butein (3,4,2',4'-Tetrahydroxychalcone), piceatannol (3,5,3 ',4'-Tetrahydroxy-trans-stilbene), isoliquiritigenin (4,2',4'-Trihydroxychalcone), fisetin (3,7,3',4'-Tetrahyddroxyflavone), quercetin (3,5,7,3',4'-Pentahydroxyflavone), Deoxyrhapontin (3,5-Dihydroxy-4'-methoxystilbene 3- ⁇ - ⁇ - D-glucoside); trans-Stilbene; Rhapontin (3,3',5-Trihydroxy-4'-methoxystilbene 3- ⁇ - ⁇ - ⁇ - glucoside); cis-Stilbene; But
  • Kaempferol (3,5,7,4'-Tetrahydroxyflavone); 6-Hydroxyapigenin (5,6,7,4'-Tetrahydoxyflavone); Scutellarein); Apigenin (5,7,4'-Trihydroxyflavone); 3,6,2',4'-Tetrahydroxyflavone; 7,4'- Dihydroxyflavone; Daidzein (7,4'-Dihydroxyisoflavone); Genistein (5,7,4'- Trihydroxyflavanone); Naringenin (5,7,4'-Trihydroxyflavanone); 3,5,7,3',4'- Pentahydroxyflavanone; Flavanone; Pelargonidin chloride (3,5,7,4'-Tetrahydroxyflavylium chloride); Hinokitiol (b-Thujaplicin; 2-hydroxy-4-isopropyl-2,4,6-cycloheptatrien-l-one); L-(+)- Ergothi
  • Ambroxol (trans-4-(2-Amino-3,5-dibromobenzylamino) cyclohexane-HCl; and U-83836E ((-)- 2-((4-(2,6-di-l-Pyrrolidinyl-4-pyrimidinyl)-l-piperzainyl)methyl)-3,4-dihydro-2,5,7,8- tetramethyl-2H-l-benzopyran-6-ol.2HCl). Analogs and derivatives thereof can also be used.
  • the sirtuin pathway activator is an AMPK pathway activator.
  • AMPK activity can be determined by any means known in the art, such as, e.g., measuring AMPK phosphorylation via an ELISA assay or by Western blot.
  • the AMPK pathway activator can be a biguanide. Examples of biguanides include and are not limited to metformin, buformin, phenformin, proguanil or the like.
  • the sirtuin pathway activator (e.g., AMPK pathway activator) is a polyphenol.
  • Exemplary polyphenols include, e.g., chlorogenic acid, resveratrol, caffeic acid, piceatannol, ellagic acid, epigallocatechin gallate (EGCG), stilbene, hydroxycinnamic acid, grape seed extract, or any analog thereof.
  • the sirtuin pathway activator is resveratrol, an analog thereof, or a metabolite thereof.
  • the activator can be pterostilbene or a small molecule analog of resveratrol. Examples of small molecule analogs of resveratrol are described in U.S. Patent Application Nos. 20070014833, 20090163476, and 20090105246, which are incorporated herein by reference in its entirety.
  • the polyphenol can be a substantially homogeneous population of polyphenols.
  • the polyphenol can be one type of polyphenol, wherein the composition can exclude all other types of polyphenols.
  • an invention method comprises administration one type of polyphenol, and exclude all other types of polyphenols.
  • an invention method comprises administration of two, three, or four types of polyphenols, and exclude all other types of polyphenols.
  • an invention method comprises
  • Sirtuin pathway activators can also include PDE inhibitors.
  • PDE inhibitors can include non-specific PDE inhibitors.
  • PDE inhibitors can be naturally occurring or no n- naturally occurring (e.g. manufactured), and may be provided in the form of a natural source comprising the PDE inhibitor, or an extract thereof (e.g. purified).
  • non-specific PDE inhibitors include, but are not limited to, caffeine, theophylline, theobromine, 3-isobutyl- l- methylxanthine (IBMX), pentoxifylline (3,7-dihydro-3,7-dimethyl-l-(5oxohexyl)-lH-purine-2, 6-dione), aminophylline, paraxanthine, and salts, derivatives, metabolites, catabolites, anabolites, precursors, and analogs thereof.
  • Non- limiting examples of natural sources of PDE inhibitors include coffee, tea, guarana, yerba mate, cocoa, and chocolate (e.g. dark chocolate).
  • Sirtuin pathway activators can include irisin, quinic acid, cinnamic acid, ferulic acid, fucoxanthin, rosiglitazone, or any analog thereof.
  • Sirtuin-pathway activators can also include isoflavones, pyroloquinoline (PQQ), quercetin, L-carnitine, lipoic acid, coenzyme Q10, pyruvate, 5-aminoimidazole-4-carboxamide ribotide (ALCAR), bezfibrate, oltipraz, and/or genistein.
  • Sirtuin pathway activators can agents that stimulate expression of the Fndc5, PGCl-a, or UCP1.
  • the expression can be measured in terms of the gene or protein expression level.
  • the sirtuin pathway activator can be irisin.
  • Methods for increasing the level of irisin are described in Bostrom et al., "A PGC1- a- dependent myokine that drives brown-fat-like development of white fat and thermogenesis," Nature, Jan 11, 2012.
  • Sirtuin pathway activators can include thiazolidinediones.
  • thiazolidinediones include, e.g. , rosiglitazone, pioglitazone, troglitazone, and any analogs thereof.
  • One or more PDE inhibitors can be used with the compositions or methods described herein, particularly the compositions including one or more NO donors and one or more branched amino acids such as leucine and/or a leucine metabolite.
  • one or more methods can further comprise administering to a subject a PDE inhibitor.
  • a PDE inhibitor can act as a sirtuin pathway activator.
  • the PDE inhibitor can be selective or nonselective.
  • the PDE inhibitor can exhibit selective inhibition to a PDE subclass, for example PDE 5. Examples of selective PDE inhibitors include inhibitors to PDE 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11.
  • a non-selective PDE inhibitor can be one that does not distinguish among sub-classes of phosphodiesterases.
  • some no n- selective PDE inhibitors may interact with more than one metabolic pathway.
  • some non-selective PDE inhibitors may be xanthine derivatives and serve as adenosine antagonists and have unknown interactions with other metabolic pathways.
  • Selective PDE inhibitors can be PDE inhibitors that exhibit preferential interaction with a selected PDE. For example, a PDE inhibitor can have a strong interaction with PDE 5, and very little interaction with other PDE sub-classes.
  • PDE inhibitors can be naturally occurring or non-naturally occurring (e.g.
  • the PDE inhibitor is a nonspecific PDE inhibitor.
  • non-specific PDE inhibitors include, but are not limited to, caffeine, theophylline, theobromine, 3-isobutyl-l- methylxanthine (IB MX), pentoxifylline (3,7- dihydro-3,7-dimethyl-l-(5oxohexyl)-lH-purine-2, 6-dione), aminophylline, paraxanthine, and salts, derivatives, metabolites, catabolites, anabolites, precursors, and analogs thereof.
  • the PDE inhibitor can be sourced from a natural source of PDE inhibitors.
  • Non- limiting examples of natural sources of PDE inhibitors include coffee, tea, guarana, yerba mate, cocoa, and chocolate (e.g. dark chocolate).
  • any agents that selectively and negatively regulate a PDE subclass, such as PDE 5, expression or activity can be used as selective PDE inhibitors in the compositions and methods described herein.
  • a selective PDE inhibitor alternatively can be an agent that exhibits a 50% inhibitory concentration (IC50) with respect to a PDE subclass, such as PDE 5, that is at least at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 1000-fold, at least 10,000-fold lower than the inhibitor's IC50 with respect to one, two, three, or more other PDE subclasses.
  • IC50 inhibitory concentration
  • a selective PDE inhibitor can be an agent that exhibits a 50% inhibitory concentration (IC50) with respect to a PDE subclass, such as PDE 5, that is at least at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 1000-fold, at least 10,000-fold, or more, lower than the inhibitor's IC50 with respect to all other PDE subclasses.
  • IC50 inhibitory concentration
  • IC50 is a determination of the concentration at which 50% of a given PDE is inhibited in a cell-based assay. IC50 determinations can be accomplished using any suitable techniques. In general, an IC50 can be determined by measuring the activity of a given enzyme in the presence of a range of concentrations of the inhibitor under study. The experimentally obtained values of enzyme activity then are plotted against the inhibitor concentrations used. The concentration of the inhibitor that shows 50% enzyme activity (as compared to the activity in the absence of any inhibitor) is taken as the "IC50" value. Analogously, other inhibitory concentrations can be defined through appropriate determinations of activity. For example, in some settings it can be desirable to establish a 90% inhibitory concentration, i.e., IC90, etc.
  • the selective PDE inhibitor may inhibit PDE activity with an IC50 value of about 100 nM or less, preferably about 50 nM, about 25 nM, about 10 nM, about 5 nM, about 1 nM, 100 pM, 50 pM, 25 pM, 10 pM, 1 pM, or less, as ascertained in a cell-based assay or an in vitro kinase assay.
  • the PDE inhibitor is a PDE1 inhibitor such as nimodipine, vinopocetine, and IC224.
  • the PDE 1 inhibitor can interact with PDE1, which is a
  • the vinopocetine can be derived from periwinkle extract, and it can serve as a
  • Vinopocetine can be in the form of a dietary supplement.
  • the PDE inhibitor is a PDE3 inhibitor such as meribendan, arinone and cilostamide.
  • the PDE inhibitor can be a PDE4 inhibitor, such as apremilast, mesembrine, ibudilast, piclamilast, luteolin, roflumilast, cilomilast, diazepam, rolipram and YM796.
  • the PDE inhibitor can be a PDE4 inhibitor, such as rolipram and YM796.
  • the PDE4 inhibitor can interact with PDE 4, which is a cAMP-specific phosphodiesterase that
  • the PDE inhibitor is a PDE5 specific inhibitor, icariin, sildenafil, tadalafil, vardenafil, avanafil, iodenafil, mirodenafil, udenafil, and zaprinast.
  • the PDE5 inhibitor is icariin.
  • the PDE5 inhibitor is sildenafil.
  • the PDE5 inhibitor is tadalafil.
  • the PDE5 inhibitor is vardenafil.
  • the PDE5 inhibitor is avanafil.
  • the PDE5 inhibitor is iodenafil.
  • the PDE5 inhibitor is mirodenafil. In some embodiments, the PDE5 inhibitor is udenafil. In some embodiments, the PDE5 inhibitor is zaprinast.
  • the PDE 5 inhibitor can interact with PDE 5, which is a cGMP- specific PDE. Increases in cGMP signaling can increase mitochondrial biogenesis both in vitro and in vivo. A PDE 5 inhibitor can increase nitric oxide signaling and be an effective vasodilator. Other examples of PDE 5 inhibitors are described in U.S. Patent No. 5,250,534 and 6,469,012, which are each incorporated by reference in their entirety.
  • a PDE inhibitor is administered in place of or in addition to resveratrol or other sirtuin pathway activator.
  • compositions comprising one or more components described herein comprise a PDE inhibitor in place of or in addition to resveratrol or other sirtuin pathway activator.
  • Certain embodiments provide methods, compositions, and kits for reducing or preventing NASH or hepatic steatosis in a subject in need thereof.
  • the invention provides a method of reducing NASH or hepatic steatosis in a subject in need thereof, comprising administering to the subject a composition comprising one or more NO donors.
  • the invention provides a method of reducing NASH or hepatic steatosis in a subject in need thereof, comprising administering to the subject a composition comprising an amount of a sirtuin pathway activator such as, e.g., metformin.
  • Another exemplary method of reducing or preventing NASH or hepatic steatosis comprises administering (a) an amount of a branched chain amino acid and/or metabolite thereof existing in form of a free amino acid, and (b) an additional agent.
  • the branched chain amino acid and/or metabolite thereof is leucine.
  • the additional agent is one or more sirtuin pathway activators or NO donors.
  • the additional agent is metformin, and/or a PDE inhibitor, e.g. sildenafil.
  • co-administration of such the branched chain amino acid and/or metabolite thereof and the additional agent reduces or prevents hepatic steatosis to a greater extent than administration of any one of the agents alone.
  • co-administration of the branched chain amino acid in free amino acid form (or metabolite thereof) and the additional agent has a synergistic effect, e.g., reduces or prevents hepatic steatosis to a greater extent than an additive effect of administering the branched chain amino acid in free amino acid form (or metabolite thereof) alone and administering the additional agent alone and can have benefits in reducing the toxicity to administer the additional agent alone.
  • the invention described herein provides methods for sustaining a reduction of nonalcoholic steatohepatitis (NASH) in a subject in need thereof.
  • the disclosed invention also provides methods for reducing liver fibrosis, reducing macrophage infiltration, reducing expression of lipogenic genes, reducing expression of hepatic inflammatory genes, and/or increasing expression of liver fatty acid oxidation genes.
  • the effectiveness of reduction of non-alcoholic steatohepatitis or hepatic steatosis can be ascertained by measuring and monitoring a level of one or more biomarkers or physiological indicators in the subject. The effectiveness of treatment using the disclosed invention can be assessed in various ways.
  • a reduction of a physiological indicator or a biomarker indicates a sustained reduction or resolution of non-alcoholic steatohepatitis (NASH) in the subject after commencement of said methods and compositions described herein.
  • NASH non-alcoholic steatohepatitis
  • method of reducing, the sustained reduction of, and/or preventing NASH or hepatic steatosis in a subject can comprise co- administering to the subject (a) one or more NO donors, and (b) a branched chain amino acid in free amino acid form and/or a metabolite thereof.
  • the branched chain amino acid in free amino acid form is leucine.
  • a metabolite of leucine is co- administered.
  • the metabolite can be, e.g., HMB or KIC.
  • a method of reducing, the sustained reduction of, and/or preventing NASH or hepatic steatosis in a subject can comprise co- administering to the subject two or more selected from the group consisting of (a) an NO donor, (b) a branched chain amino acid in free amino acid form or metabolite thereof, and optionally (c) a sirtuin pathway activator, and optionally (d) a PDE inhibitor.
  • the NO donor is L-arginine and/or sodium nitroprusside.
  • the sirtuin pathway activator is an AMPK activator.
  • the AMPK activator is a biguanide.
  • the biguanide is metformin.
  • the branched chain amino acid in free amino acid form is leucine.
  • the PDE inhibitor is a PDE5 inhibitor.
  • Exemplary PDE5 inhibitors are described herein.
  • the PDE5 inhibitor can be, for example, icariin.
  • the PDE5 inhibitor can be any one of sildenafil, tadalafil, vardenafil, udenafil, or zaprinast.
  • metformin is co- administered with L-arginine and/or SNP and a metabolite of leucine.
  • the metabolite of leucine is HMB.
  • the metabolite of leucine is KIC.
  • more than one NO donors and/or sirtuin pathway activators are co -administered with a branched chain amino acid in free amino acid form or metabolite thereof (e.g., co -administered with leucine in free amino acid form, HMB, or KIC).
  • a method may comprise co- administering an NO donor, a biguanide, a polyphenol, and a branched chain amino acid in free amino acid form or metabolite thereof.
  • a method can comprise co- administering to a subject L-arginine and/or SNP, metformin, resveratrol, and leucine in free amino acid form.
  • Certain embodiments can comprise co- administering to a subject L-arginine and/or SNP, metformin, resveratrol, and HMB.
  • a method can comprise coadministering to a subject L-arginine and/or SNP, metformin, resveratrol, and KIC.
  • a method can comprise co- administering to a subject L-arginine and/or SNP, metformin, leucine, and sildenafil.
  • Another method of reducing, the sustained reduction of, and/or preventing NASH or hepatic steatosis in a subject can comprise co- administering to the subject (a) one or more NO donors, (b) a branched chain amino acid in free amino acid form and/or a metabolite thereof and(c) a PDE inhibitor.
  • the PDE inhibitor is a PDE5 inhibitor.
  • the PDE5 inhibitor can be, for example, icariin.
  • the PDE5 inhibitor can be any one of sildenafil, tadalafil, vardenafil, udenafil, or zaprinast.
  • the particular choice of agents depends upon the diagnosis of the attending physicians and their judgment of the condition of the individual and the appropriate treatment protocol.
  • the agents are optionally administered concurrently (e.g., simultaneously, essentially simultaneously or within the same treatment protocol) or sequentially, depending upon the nature of the disease, disorder, or condition, the condition of the individual, and the actual choice of compounds used.
  • the determination of the order of administration, and the number of repetitions of administration of each therapeutic agent during a treatment protocol is based on an evaluation of the disease being treated and the condition of the individual.
  • therapeutically-effective dosages vary when the drugs are used in treatment combinations. Any suitable methods for experimentally determining therapeutically- effective dosages of drugs and other agents for use in combination treatment regimens can be used.
  • dosages of the co -administered agents vary depending on the type of co-drug employed, on the specific drug employed, on the disease or condition being treated and so forth.
  • the agents provided herein is optionally administered either simultaneously with the biologically active agent(s), or sequentially. In certain instances, if administered sequentially, the attending physician will decide on the appropriate sequence of therapeutic compound described herein in combination with the additional therapeutic agent.
  • the multiple therapeutic agents are optionally administered in any order or even simultaneously.
  • the therapeutic agents are optionally provided in a single, unified form, or in multiple forms (by way of example only, either as a single pill or as two separate pills). In certain instances, one of the therapeutic agents is optionally given in multiple doses. In other instances, both are optionally given as multiple doses. If not simultaneous, the timing between the multiple doses is any suitable timing, e.g., from more than zero weeks to less than four weeks. In addition, the combination methods, compositions and formulations are not to be limited to the use of only two agents; the use of multiple therapeutic combinations is also envisioned (including two or more compounds described herein).
  • a dosage regimen to treat, prevent, or ameliorate the condition(s) for which relief is sought is modified in accordance with a variety of factors. These factors include the disorder from which the subject suffers, as well as the age, weight, sex, diet, and medical condition of the subject. Thus, in various embodiments, the dosage regimen actually employed varies and deviates from the dosage regimens set forth herein.
  • the pharmaceutical agents that make up the combination therapy described herein are provided in a combined dosage form or in separate dosage forms intended for substantially simultaneous administration.
  • the pharmaceutical agents that make up the combination therapy described herein are provided in a combined dosage form or in separate dosage forms intended for substantially simultaneous administration.
  • two-step administration regimen calls for sequential administration of the active agents or spaced-apart administration of the separate active agents.
  • the time period between the multiple administration steps varies, by way of non- limiting example, from a few minutes to several hours, depending upon the properties of each pharmaceutical agent, such as potency, solubility, bioavailability, plasma half-life and kinetic profile of the pharmaceutical agent.
  • compositions for use in practicing one or more methods are formulated such that they do not contain (or exclude) one or more of the following ingredients: caffeine, green tea extract or extracts from guarana seed or guarana plants.
  • Compositions can also be formulated such that they are substantially free of high glycemic index carbohydrate, such as, e.g., simple carbohydrates, including sugars such as but not limited to sucrose, glucose, dextrose, maltose, fructose, and galactose, among others.
  • carbohydrate such as, e.g., simple carbohydrates, including sugars such as but not limited to sucrose, glucose, dextrose, maltose, fructose, and galactose, among others.
  • the amount of pharmaceutical agent, or any other component used in a combination composition described herein can be an amount that is therapeutically effective.
  • the amount of a pharmaceutical agent, or any other component used in a combination composition described herein can be an amount that is sub-therapeutic.
  • using sub-therapeutic amounts of an agent or component can reduce the side-effects of the agent. Use of subtherapeutic amounts can still be effective, particularly when used in synergy with other agents or components.
  • a sub-therapeutic amount of the agent or component can be such that it is an amount below which would be considered therapeutic.
  • FDA guidelines can suggest a specified level of dosing to treat a particular condition, and a sub-therapeutic amount would be any level that is below the FDA suggested dosing level.
  • the sub-therapeutic amount can be about 1, 5, 10, 15, 20, 25, 30, 35, 50, 75, 90, or 95% less than the amount that is considered to be a therapeutic amount.
  • the therapeutic amount can be assessed, assayed, interrogated or analyzed for individual subjects, or for groups of subjects.
  • the group of subjects can be all potential subjects, or subjects having a particular characteristic such as age, weight, race, gender, or physical activity level.
  • a physician suggested starting dose can be 1000 mg daily, with subject specific dosing having a range of 500 mg to a maximum of 2550 mg daily
  • the particular dosing for a subject can be determined by a clinician by titrating the dose and measuring the therapeutic response.
  • the therapeutic dosing level can be determined by measuring fasting plasma glucose levels and measuring glycosylated hemoglobin.
  • a sub-therapeutic amount can be any level that would be below the recommended dosing of metformin. For example, if a subject's therapeutic dosing level is determined to be 700 mg daily, a dose of 600 mg would be a sub-therapeutic amount. Alternatively, a sub-therapeutic amount can be determined relative to a group of subjects rather than an individual subject.
  • a sub-therapeutic amount can be any amount below 2000 mg.
  • the dosing can be recommended by a healthcare provider including, but not limited to a patient's physician, nurse, nutritionist, pharmacist, or other health care professional.
  • a health care professional may include a person or entity that is associated with the health care system. Examples of health care professionals may include surgeons, dentists, audio logists, speech pathologists, physicians (including general practitioners and specialists), physician assistants, nurses, midwives,
  • compositions can comprise any combination of agents, like NO donors, leucine, metabolites of leucine, such as HMB or KIC, PDE inhibitors (e.g., PDE5 inhibitors such as, by way of example only, sildenafil, icariin), sirtuin pathway activators such as, e.g., AMPK activators, polyphenols such as, e.g., resveratrol, and sirtuin activators, that have been isolated from one or more sources.
  • PDE inhibitors e.g., PDE5 inhibitors such as, by way of example only, sildenafil, icariin
  • sirtuin pathway activators such as, e.g., AMPK activators
  • polyphenols such as, e.g., resveratrol
  • sirtuin activators that have been isolated from one or more sources.
  • the agents can be isolated from natural sources or created from synthetic sources and then enriched to increase the purity of the components
  • sildenafil can be created from a synthetic source and then enriched by one or more purification methods.
  • leucine e.g., free leucine
  • the isolated and enriched components such as, e.g., metformin, resveratrol, icariin, sildenafil, free leucine, HMB, and KIC can then be formulated for administration to a subject in any
  • Any of the methods described herein can comprise administering an effective dose of a NO donors, e.g., L-arginine and/or SNP.
  • the dose of NO donors can be a therapeutic dose or a sub-therapeutic dose.
  • the dose of a NO donor can be about, less than about, or more than about 0.01 mg, 0.02 mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 20mg, 25mg, 50mg, 75mg, lOOmg, 125mg, 145mg, 150mg, 175mg, 200mg, 225mg, 250mg, 300mg, 350mg, 500mg, lg, 2g, 3g, 4g, 5g, 6g, 7g, 8g, 9g, 10 g (per administration or per day) or any
  • the dose of L-arginine can be about, less than about, or more than about 0.01 mg, 0.02 mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 20mg, 25mg, 50mg, 75mg, lOOmg, 125mg, 145mg, 150mg, 175mg, 200mg, 225mg, 250mg, 300mg, 350mg, 500mg, lg, 2g, 3g, 4g, 5g, 6g, 7g, 8g, 9g, 10 g (per administration or per day) or any
  • the dose of SNP can be about, less than about, or more than about 0.01 mg, 0.02 mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg (per administration or per day) or any intermediate numbers or ranges.
  • the dose of an organic nitrate can be about, less than about, or more than about 0.01 mg, 0.02 mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 20mg, 25mg, 50mg, 75mg, lOOmg, 125mg, 145mg, 150mg, 175mg, 200mg, 225mg, 250mg, 300mg, 350mg, 500mg (per administration or per day) or any intermediate numbers or ranges.
  • the dose of a diazeniumdiolate (NONOate) can be about, less than about, or more than about 0.01 mg, 0.02 mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 20mg, 25mg, 50mg, 75mg, lOOmg, 125mg, 145mg, 150mg, 175mg, 200mg, 225mg, 250mg, 300mg, 350mg, 500mg (per administration or per day) or any intermediate numbers or ranges.
  • NONOate diazeniumdiolate
  • the dose of a S-nitrosothiol can be about, less than about, or more than about 0.01 mg, 0.02 mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 20mg, 25mg, 50mg, 75mg, lOOmg, 125mg, 145mg, 150mg, 175mg, 200mg, 225mg, 250mg, 300mg, 350mg, 500mg (per administration or per day) or any intermediate numbers or ranges.
  • the dose of a pharmaceutical agent comprising one or more NO groups can be about, less than about, or more than about 0.01 mg, 0.02 mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 20mg, 25mg, 50mg, 75mg, lOOmg, 125mg, 145mg, 150mg, 175mg, 200mg, 225mg, 250mg, 300mg, 350mg, 500mg, lg, 2g, 3g, 4g, 5g, 6g, 7g, 8g, 9g, 10 g (per administration or per day) or any intermediate numbers or ranges.
  • the dose can be administered daily.
  • the dose can be a low dose, a medium dose, or a high dose.
  • a low dose of L-arginine may comprise about, less than about, or more than about 20mg, 25mg, 50mg, 75mg, lOOmg, 125mg, 145mg, or more (or any intermediate numbers or ranges);
  • a medium dose of L-arginine may comprise about, less than about, or more than about 150mg, 175mg, 200mg, 225mg, 250mg, 300mg, 350mg, 500mg, or more (or any intermediate numbers or ranges); and
  • a high dose of L-arginine may comprise about, less than about or more than about 600 mg, 700 mg.
  • a daily low dose of L-arginine may comprise about, less than about, or more than about 20mg, 25mg, 50mg, 75mg, lOOmg, 125mg, 140mg, or more (or any intermediate numbers or ranges);
  • a daily medium dose of L-arginine may comprise about, less than about, or more than about 150mg, 175mg, 200mg, 225mg, 250mg, 300mg, 350mg, 500mg, or more (or any intermediate numbers or ranges); and
  • a daily high dose of L-arginine may comprise about, less than about or more than about 600 mg, 700 mg. 800 mg, 900 mg, lg, 2g, 3g, 4g, 5g, 6g, 7g, 8g, 9g, 10 g or more (or any intermediate numbers
  • a low dose of SNP may comprise about, less than about, or more than about 0.01 mg, 0.02 mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 1 mg, or more (or any intermediate numbers or ranges);
  • a medium dose of SNP may comprise about, less than about, or more than about 2 mg, 3 mg, 4 mg, 5mg, 6 mg, 7 mg, 8 mg, or more (or any intermediate numbers or ranges); and
  • a high dose of SNP may comprise about, less than about, or more than about 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, or more.
  • a daily low dose of SNP may comprise about, less than about, or more than about 0.01 mg, 0.02 mg, 0.03 mg,
  • a daily medium dose of SNP may comprise about, less than about, or more than about 2 mg, 3 mg, 4 mg, 5mg, 6 mg, 7 mg, 8 mg, or more (or any intermediate numbers or ranges); and a daily high dose of L-arginine may comprise about, less than about, or more than about 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, or more (or any intermediate numbers or ranges).
  • Any of the methods described herein can comprise administering a dose of a branched chain amino acid in free amino acid form, and/or a dose of a metabolite thereof.
  • the dose of the branched chain amino acid in free amino acid form, or metabolite thereof can be a therapeutic dose.
  • the dose of the branched chain amino acid in free amino acid form or metabolite thereof can be a sub-therapeutic dose.
  • a sub-therapeutic dose of leucine in free amino acid form can be about, less than about, or more than about 0.25 - 3.0 g (e.g. 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.5, 3, or more g).
  • a sub-therapeutic dose of leucine in free amino acid form can be about, less than about, or more than about 0.25 - 3.0 g/day (e.g. 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.5, 3, or more g/day).
  • the method comprises administering less than 3.0 g leucine in free amino acid form per day.
  • a sub-therapeutic dose of HMB can be about, less than about, or more than about 0.05 - 3.0 g (e.g. 0.05, 0.1, 0.2, 0.4, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, or more g).
  • a sub-therapeutic dose of HMB can be about, less than about, or more than about 0.05 - 3.0 g/day (e.g. 0.05, 0.1, 0.2, 0.4, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, or more g/day).
  • a sub-therapeutic dose of KIC can be about, less than about, or more than about 0.1 - 3.0 g (e.g. 0.1, 0.2, 0.4, 0.5, 0.75,
  • a sub-therapeutic dose of KIC can be about, less than about, or more than about 0.1 - 3.0 g/day (e.g. 0.1, 0.2, 0.4, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.5, 3, or more g/day).
  • any of the methods described herein can comprise administering a dose of sirtuin pathway activators.
  • the sirtuin pathway activator is a sirtuin activator.
  • the sirtuin activator is resveratrol.
  • the sirtuin activator is a polyphenol selected from the group consisting of chlorogenic acid, resveratrol, caffeic acid, cinnamic acid, ferulic acid, piceatannol, ellagic acid, epigallocatechin gallate, grape seed extract, and any analog thereof.
  • the sirtuin pathway activator is an AMPK activator.
  • AMPK activator is a biguanide.
  • the biguanide is metformin.
  • the dose of a sirtuin pathway activator can be about, less than about, or more than about 0.01 mg, 0.02 mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 20mg, 25mg, 50mg, 75mg, lOOmg, 125mg, 145mg, 150mg, 175mg, 200mg, 225mg, 250mg, 300mg, 350mg, 500mg, lg, 2g, 3g, 4g, 5g, 6g, 7g, 8g, 9g, 10 g, 20 g, 30 g, 40 g, 50 g, 60 g, 70 g, 80 g, 90 g, or
  • Any of the methods described herein can comprise administering a dose of metformin.
  • the dose of metformin can be a therapeutic dose.
  • the therapeutic dose can be above 500 mg, above 600 mg, above 700 mg, above 800 mg, above 900 mg, or above 1000 mg (1 g).
  • a therapeutic dose of metformin can be about 1 g, about 1.1 g, about 1.2 g, about 1.3 g, about 1.4 g, about 1.5 g, about 1.6 g, about 1.7 g, about 1.8 g, about 1.9 g, about 2 g, or more than 2 g.
  • the therapeutic dose can be above 500 mg/day, above 600 mg/day, above 700 mg/day, above 800 mg/day, above 900 mg/day, above 1000 mg/day (1 g/day).
  • a therapeutic dose of metformin can be about 1 g/day, about 1.1 g/day, about 1.2 g/day, about 1.3 g/day, about 1.4 g/day, about 1.5 g/day, about 1.6 g/day, about 1.7 g/day, about 1.8 g/day, about 1.9 g/day, about 2 g/day, or more than 2 g/day.
  • the dose of metformin can be a sub-therapeutic dose.
  • the sub-therapeutic dose of metformin can be about 1-1000 mg, about 5-500 mg, about 10-100 mg, about 30-90 mg, about 50-70 mg, or about 62.5 mg.
  • the sub-therapeutic dose of metformin can be, e.g., 1, 5, 10, 15, 25, 50, 125, 250, or 500 mg.
  • the dose of metformin can be a sub-therapeutic dose.
  • the subtherapeutic dose of metformin can be about 40-70, 25-1000 mg/day, about 50-500 mg/day, about 100-500 mg/day.
  • the sub-therapeutic dose of metformin can be about, less than about, or greater than about 1, 5, 10, 15, 25, 50, 60 125, 250, or 500 mg/day.
  • the metformin can be administered as a unit dose.
  • the unit dose of metformin can be 1, 5, 10, 15, 25, 50, 62.5, 125 or 250 mg.
  • Any of the methods described herein can comprise administering a dose of a polyphenol, e.g., resveratrol.
  • the dose may be administered daily.
  • the dose can be a low dose, a medium dose, or a high dose.
  • a low dose of resveratrol may comprise about, less than about, or more than about 0.5 mg, 1 mg, 2.5mg, 5mg, 7.5mg, lOmg, 12.5mg, 15mg, 20mg, 25mg, 50mg, 75mg, lOOmg, or more;
  • a medium dose of resveratrol may comprise about, less than about, or more than about 20mg, 25mg, 50mg, 75mg, lOOmg, 125mg, 150mg, 175mg, 200mg, 250mg, or more;
  • a high dose of resveratrol may comprise about, less than about, or more than about 150mg, 175mg, 200mg, 225mg, 250mg, 300mg, 350mg, 400mg, or more.
  • a daily low dose of resveratrol may comprise about, less than about, or more than about 0.5mg, 1 mg, 2.5mg, 5mg, 7.5mg, lOmg, 12.5mg, 15mg, 20mg, 25mg, 50mg, 75mg, lOOmg, or more;
  • a daily medium dose of resveratrol may comprise about, less than about, or more than about 20mg, 25mg, 50mg, 75mg, lOOmg, 125mg, 150mg, 175mg, 200mg, 250mg, or more;
  • a daily high dose of resveratrol may comprise about, less than about, or more than about 150mg, 175mg, 200mg, 225mg, 250mg, 300mg, 350mg, 400mg, or more.
  • Any of described herein can comprise administering a dose of a thiazolidinedione.
  • exemplary thiazoladinediones are described herein.
  • the dose of thiazolidinedione can be a therapeutic dose or a sub-therapeutic dose.
  • the thiazolidinedione can be rosiglitazone.
  • the dose of the rosiglitazone can be at least 100 ⁇ g.
  • the dose of the rosiglitazone can be about or less than about 4 mg.
  • the dose of the rosiglitazone can be 100 ⁇ g - 4 mg, can be 200 ⁇ g - 2 mg, can be 400 ⁇ g to 1000 ⁇ g.
  • the thiazolidinedione can be pioglitazone.
  • the dose of the pioglitazone can be at least 100 ⁇ g.
  • the dose of the pioglitazone can be about or less than about 15 mg.
  • the dose of the pioglitazone can be 100 ⁇ g - 45 mg, can be 200 ⁇ g - 10 mg, can be 400 ⁇ g to 5 mg, can be 500 ⁇ g to 1 mg.
  • a composition comprises an amount of an NO donor and a branched amino acid such as leucine or a leucine metabolite, and optionally a selective PDE inhibitor (e.g., PDE-5 inhibitor including but not limited to sildenafil or icariin).
  • a composition comprising an amount of an NO donor and a branched amino acid such as leucine or a leucine metabolite can optionally exclude the use of a selective PDE inhibitor.
  • the amount of a PDE inhibitor may be a sub-therapeutic amount, and/or an amount that is synergistic with one or more other compounds in the composition or one or more of the compounds administered simultaneously or in close temporal proximity with the composition.
  • the PDE inhibitor is administered in a low dose, a medium dose, or a high dose, which describes the relationship between two doses, and generally do not define any particular dose range.
  • a dose of sildenafil can be about or less than about 0.05, 0.1, 0.5, 1, 2, 5, 10, 20, 40, 60, 80, or 100 mg of sildenafil.
  • a dose of sildenafil can be about 0.05-100, 1-50, or 5-20 mg of sildenafil.
  • a dose of icariin can be about or less than about 1, 10, 12.5, 20, 25, 50, 100, 150, 300, 400, 500, 750, 1000, 1500, or 2000 mg of icariin.
  • a dose of icariin can be about 1-100, 5-60, or 10-30 mg of icariin.
  • a dose of tadalafil can be about 0.01, 0.05, 0.1, 0.5, 1, 15, 2, 2.5, 5, 10, 15, or 20 mg.
  • a dose of tadalafil can be about 0.1-50, 0.5-20, or 1-10 mg.
  • a dose of vardenafil can be about 0.01, 0.05, 0.1, 0.5, 1, 15, 2, 2.5, 5, 10, 15, or 20 mg.
  • a dose of vardenafil can be about 0.1-50, 0.5-20, or 1-10 mg.
  • a dose of avanafil can be about 1, 10, 20, 50, 100, 150, 300, 400, 500, 750, 1000, 1500, or 2000 mg.
  • a dose of avanafil can be about 1-100, 5-50, or 10-40 mg.
  • a dose of lodenafil can be about 0.05, 0.1, 0.5, 1, 2, 5, 10, 20, 50, 80, 100, 150, 300, 400, 500, 750, 1000, 1500, or 2000 mg.
  • a dose of lodenafil can be about 0.05-100, 1-50, or 5-20 mg.
  • a dose of mirodenafil can be about 0.05, 0.1, 0.5, 1, 2, 5, 10, 20, 50, 80, 100, 150, 300, 400, 500, 750, 1000, 1500, or 2000 mg.
  • a dose of mirodenafil can be about 0.05-100, 1-50, or 5-20 mg.
  • a dose of udenafil can be about 0.05, 0.1, 0.5, 1, 2, 5, 10, 20, 50, 80, 100, 150, 300, 400, 500, 750, 1000, 1500, or 2000 mg.
  • a dose of udenafil can be about 0.05-100, 1-50, or 5-20 mg.
  • a daily dose of sildenafil can be about or less than about 0.05, 0.1, 0.5, 1, 2, 5, 10, 20, 40, 60, 80, or 100 mg of sildenafil.
  • a daily dose of sildenafil can be about 0.05-100, 1-50, or 5- 20 mg of sildenafil.
  • a daily dose of icariin can be about or less than about 1, 10, 20, 25, 50, 100, 150, 300, 400, 500, 750, 1000, 1500, or 2000 mg of icariin.
  • a daily dose of icariin can be about 1-100, 10-75, or 20-40 mg of icariin.
  • a daily dose of tadalafil can be about 0.01, 0.05, 0.1, 0.5, 1, 15, 2, 2.5, 5, 10, 15, or 20 mg.
  • a daily dose of tadalafil can be about 0.1-50, 0.5-20, or 1-10 mg.
  • a daily dose of vardenafil can be about 0.01, 0.05, 0.1, 0.5, 1, 15, 2, 2.5, 5, 10, 15, or 20 mg.
  • a daily dose of vardenafil can be about 0.1-50, 0.5-20, or 1-10 mg.
  • a daily dose of avanafil can be about 1, 10, 20, 50, 100, 150, 300, 400, 500, 750, 1000, 1500, or 2000 mg.
  • a daily dose of avanafil can be about 1-100, 5-50, or 10-40 mg.
  • a daily dose of lodenafil can be about 0.05, 0.1, 0.5, 1, 2, 5, 10, 20, 50, 80, 100, 150, 300, 400, 500, 750, 1000, 1500, or 2000 mg.
  • a daily dose of lodenafil can be about 0.05-100, 1-50, or 5-20 mg.
  • a daily dose of mirodenafil can be about 0.05, 0.1, 0.5, 1, 2, 5, 10, 20, 50, 80, 100, 150, 300, 400, 500, 750, 1000, 1500, or 2000 mg.
  • a daily dose of mirodenafil can be about 0.05-100, 1-50, or 5-20 mg.
  • a daily dose of udenafil can be about 0.05, 0.1, 0.5, 1, 2, 5, 10, 20, 50, 80, 100, 150, 300, 400, 500, 750, 1000, 1500, or 2000 mg.
  • a daily dose of udenafil can be about 0.05-100, 1-50, or 5-20 mg.
  • a dose of sildenafil can be about or less than about 4 mg. In some embodiments, a daily dose of sildenafil can be about or less than about 4 mg.
  • compositions comprising synergizing amounts of PDE-5 inhibitor, such as, sildenafil, icariin, tadalafil, vardenafil, avanafil, lodenafil, mirodenafil and udenafil, in combination with leucine, HMB, KIC, and/or resveratrol.
  • PDE-5 inhibitor such as, sildenafil, icariin, tadalafil, vardenafil, avanafil, lodenafil, mirodenafil and udenafil
  • PDE-5 inhibitor such as, sildenafil, icariin, tadalafil, vardenafil, avanafil, lodenafil, mirodenafil and udenafil
  • PDE-5 inhibitor such as, sildenafil, icariin, tadalafil, vardenafil, avanafil, lodenafil, mirodenafil and u
  • compositions comprising leucine in an amount of about 0.25 - about 3.0 g, HMB in an amount of about 0.10 to about 3.0 g and sildenafil in an amount of about 0.05 - 100 mg.
  • the PDE inhibitor may be provided in an amount that produces a peak plasma concentration of about, less than about, or more than about 0.1, 1, 5, 10, 25, 50, 100, 500, 1000, 2500, 5000, 10000, or more nM.
  • Certain embodiments provide a method that comprises co- administering any of the compositions described herein.
  • Certain embodiment provide a composition comprising two or more of the following: (a) a leucine or leucine metabolite, such as HMB or KIC, present at an amount of about, less than about, or more than about 100 mg, 125mg, 150mg, 175mg, 200mg, 225mg, 250mg, 300mg, 350mg, 500mg, lg, 2g, 3g, 4g, 5g, 6g, 7g, 8g, 9g, 10 g (per
  • composition may further comprise (c) an anti-diabetic agent, such as a biguanide like metformin or a thiazoladinedione like rosiglitazone, present at an amount of about, less than about, or more than about 1 mg, 2 mg, 3 mg, 4 mg, 5mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 20mg, 25mg, 50mg, 75mg, lOOmg, 125mg, 145mg, 150mg, 175mg, 200mg, 225mg, 250mg, 300mg, 350mg, 500mg, lg, 2g, 3g, 4g, 5g, 6g, 7g, 8g, 9g, 10 g (per administration or per day) or any intermediate numbers or ranges.
  • an anti-diabetic agent such as a biguanide like metformin or a thiazol
  • the composition may further comprise (d) one or more of sirtuin pathway activator(s), which may be a PDE inhibitor, PDE 5 inhibitor, and/or a polyphenol, such as sildenafil or resveratrol, present_at an amount of about, less than about, or more than about 0.01 mg, 0.02 mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg (per administration or per day) or any intermediate numbers or ranges.
  • sirtuin pathway activator(s) which may be a PDE inhibitor, PDE 5 inhibitor, and/or a polyphenol, such as sildenafil or resveratrol, present_at an amount of about, less than about, or more than about 0.01 mg, 0.02 mg, 0.03 mg, 0.04 mg, 0.05 mg, 0.1 mg, 0.2 mg
  • Certain embodiments provide a method that comprises co- administering leucine in free amino acid form in an amount of about 0.25 to about 3.0 g/day and L-arginine in an amount of about 100 mg/day to about 10 g/day or SNP in an amount of about 0.1 mg/day to about 10 mg/day.
  • Other embodiments comprise co- administering HMB in an amount of 0.10 - 3.0 g/day and L-arginine in an amount of about 100 mg/day to about 10 g/day or SNP in an amount of about 0.0.05 mg/day to about 10 mg/day.
  • Other embodiments comprise co- administering KIC in an amount of 0.20 - 3.0 g/day and L-arginine in an amount of about 100 mg/day to about 10 g/day or SNP in an amount of about 0.05 mg/day to about 10 mg/day.
  • Other embodiments comprise co- administering leucine in free amino acid form in an amount of about 0.5 - about 3.0 g/day, HMB in an amount of about 0.10 to about 3.0 g/day and L-arginine in an amount of about 100 mg/day to about 10 g/day or SNP in an amount of about 0.05 mg/day to about 10 mg/day.
  • inventions comprise co- administering leucine in free amino acid form in an amount of about 0.5 - about 3.0 g/day, KIC in an amount of about 0.20 to about 3.0 g/day and L-arginine in an amount of about 100 mg/day to about 10 g/day or SNP in an amount of about 0.1 mg/day to about 10 mg/day.
  • inventions comprise co- administering leucine in free amino acid form in an amount of about 0.25 - about 3.0 g/day, HMB in an amount of about 0.10 to about 3.0 g/day, KIC in an amount of about 0.20 to about 3.0 g/day, and L-arginine in an amount of about 100 mg/day to about 10 g/day or SNP in an amount of about 0.05 mg/day to about 10 mg/day.
  • Some embodiments comprise co- administering leucine in free amino acid form in an amount of about 0.25 to about 3.0 g/day, metformin in an amount of about 25 to about 500 mg/day, and L-arginine in an amount of about 100 mg/day to about 10 g/day or SNP in an amount of about 0.1 mg/day to about 10 mg/day.
  • Other embodiments comprise co- administering HMB in an amount of 0.10 - 3.0 g/day, metformin in an amount of about 25 - 500 mg/day, and L-arginine in an amount of about 100 mg/day to about 10 g/day or SNP in an amount of about 0.05 mg/day to about 10 mg/day.
  • Other embodiments comprise co- administering KIC in an amount of 0.20 - 3.0 g/day, metformin in an amount of about 25 - 500 mg/day, and L-arginine in an amount of about 100 mg/day to about 10 g/day or SNP in an amount of about 0.05 mg/day to about 10 mg/day.
  • Other embodiments comprise co- administering leucine in free amino acid form in an amount of about 0.25 - about 3.0 g/day, HMB in an amount of about 0.40 to about 3.0 g/day, metformin in an amount of about 25 to about 500 mg/day, and L-arginine in an amount of about 100 mg/day to about 10 g/day or SNP in an amount of about 0.05 mg/day to about 10 mg/day.
  • inventions comprise co- administering leucine in free amino acid form in an amount of about 0.25 - about 3.0 g/day, KIC in an amount of about 0.20 to about 3.0 g/day, metformin in an amount of about 25 to about 500 mg/day, and L-arginine in an amount of about 100 mg/day to about 10 g/day or SNP in an amount of about 0.05 mg/day to about 10 mg/day.
  • Other embodiments comprise co- administering leucine in free amino acid form in an amount of about 0.25 - about 3.0 g/day, HMB in an amount of about 0.10 to about 3.0 g/day, KIC in an amount of about 0.20 to about 3.0 g/day, metformin in an amount of about 25 to about 500 mg/day, and L-arginine in an amount of about 100 mg/day to about 10 g/day or SNP in an amount of about 0.05 mg/day to about 10 mg/day.
  • Some embodiments comprise co- administering leucine in free amino acid form in an amount of about 0.25 to about 3.0 g/day, a PDE5 inhibitor in an amount of about 0.05 to about 2000 mg/day, and L-arginine in an amount of about 100 mg/day to about 10 g/day or SNP in an amount of about 0.05 mg/day to about 10 mg/day.
  • Other embodiments comprise coadministering HMB in an amount of 0.10 - 3.0 g/day, a PDE5 inhibitor in an amount of about 0.05 to about 2000 mg/day, and L-arginine in an amount of about 100 mg/day to about 10 g/day or SNP in an amount of about 0.05 mg/day to about 10 mg/day.
  • Other embodiments comprise co -administering KIC in an amount of 0.20 - 3.0 g/day, a PDE5 inhibitor in an amount of about 0.05 to about 2000 mg/day, and L-arginine in an amount of about 100 mg/day to about 10 g/day or SNP in an amount of about 0.05 mg/day to about 10 mg/day.
  • inventions comprise co -administering leucine in free amino acid form in an amount of about 0.25 - about 3.0 g/day, HMB in an amount of about 0.40 to about 3.0 g/day, a PDE5 inhibitor in an amount of about 0.05 to about 2000 mg/day, and L-arginine in an amount of about 100 mg/day to about 10 g/day or SNP in an amount of about 0.05 mg/day to about 10 mg/day.
  • inventions comprise co -administering leucine in free amino acid form in an amount of about 0.25 - about 3.0 g/day, KIC in an amount of about 0.20 to about 3.0 g/day, a PDE5 inhibitor in an amount of about 0.05 to about 2000 mg/day, and L-arginine in an amount of about 100 mg/day to about 10 g/day or SNP in an amount of about 0.05 mg/day to about 10 mg/day.
  • Other embodiments comprise coadministering leucine in free amino acid form in an amount of about 0.25 - about 3.0 g/day, HMB in an amount of about 0.10 to about 3.0 g/day, KIC in an amount of about 0.20 to about 3.0 g/day, a PDE5 inhibitor in an amount of about 0.05 to about 2000 mg/day, and L-arginine in an amount of about 100 mg/day to about 10 g/day or SNP in an amount of about 0.05 mg/day to about 10 mg/day.
  • the PDE5 inhibitor can be any of the PDE5 inhibitors described herein.
  • the daily dose of the PDE5 inhibitor can be as described herein.
  • such methods further comprise co- administering L-arginine in an amount of about 100 mg/day to about 10 g/day or SNP in an amount of about 0.05 mg/day to about 10 mg/day.
  • such methods further comprise co- administering resveratrol in an amount of about 50 to about 500 mg/day or co-administering metformin in an amount of about 25 and about 500 mg/day.
  • Some embodiments comprise co- administering leucine in free amino acid form in an amount of about 0.25 to about 3.0 g/day, metformin in an amount of about 25 to about 500 mg/day, a PDE5 inhibitor in an amount of about 0.05 to about 2000 mg/day, and in some embodiments, such methods further comprise co- administering L-arginine in an amount of about 100 mg/day to about 10 g/day or SNP in an amount of about 0.05 mg/day to about 10 mg/day.
  • the PDE5 inhibitor is sildenafil.
  • HMB in an amount of 0.10 - 3.0 g/day
  • metformin in an amount of about 25 - 500 mg/day
  • a PDE5 inhibitor e.g. sildenafil
  • L-arginine in an amount of about 100 mg/day to about 10 g/day or SNP in an amount of about 0.05 mg/day to about 10 mg/day
  • KIC in an amount of 0.20 - 3.0 g/day
  • metformin in an amount of about 25 - 500 mg/day
  • PDE5 inhibitor e.g.
  • sildenafil in an amount of about 0.05 to about 2000 mg/day, and L-arginine in an amount of about 100 mg/day to about 10 g/day or SNP in an amount of about 0.05 mg/day to about 10 mg/day.
  • Other embodiments comprise co- administering leucine in free amino acid form in an amount of about 0.25 - about 3.0 g/day, HMB in an amount of about 0.40 to about 3.0 g/day, metformin in an amount of about 25 to about 500 mg/day, a PDE5 inhibitor, e.g.
  • sildenafil in an amount of about 0.05 to about 2000 mg/day, and L-arginine in an amount of about 100 mg/day to about 10 g/day or SNP in an amount of about 0.05 mg/day to about 10 mg/day.
  • Other embodiments comprise co- administering leucine in free amino acid form in an amount of about 0.25 - about 3.0 g/day, KIC in an amount of about 0.20 to about 3.0 g/day, metformin in an amount of about 25 to about 500 mg/day, a PDE5 inhibitor, e.g.
  • sildenafil in an amount of about 0.05 to about 2000 mg/day
  • L-arginine in an amount of about 100 mg/day to about 10 g/day or SNP in an amount of about 0.05 mg/day to about 10 mg/day.
  • Other embodiments comprise co- administering leucine in free amino acid form in an amount of about 0.25 - about 3.0 g/day, HMB in an amount of about 0.10 to about 3.0 g/day, KIC in an amount of about 0.20 to about 3.0 g/day, metformin in an amount of about 25 to about 500 mg/day, a PDE5 inhibitor, e.g.
  • such methods further comprise co- administering resveratrol in an amount of about 50 to about 500 mg/day.
  • any of the above agents can be administered in unit doses. Any of the above agents in the amounts described herein can be administered in unit doses.
  • a unit dose can be a fraction of the daily dose, such as the daily dose divided by the number of unit doses to be administered per day.
  • a unit dose can be a fraction of the daily dose that is the daily dose divided by the number of unit doses to be administered per day and further divided by the number of unit doses (e.g. tablets) per administration.
  • the number of unit doses per administration may be about, less than about, or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.
  • the number of doses per day may be about, less than about, or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.
  • the number of unit doses per day may be determined by dividing the daily dose by the unit dose, and may be about, less than about, or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, 20, or more unit doses per day.
  • a unit dose can be about the daily dose or about 1/2, 1/3, 1/4, 1/5, 1/6, 1/7, 1/8, 1/9, 1/10 of the daily dose.
  • a unit dose can be about one-third of the daily amount and administered to the subject three times daily.
  • a unit dose can be about one- half of the daily amount and administered to the subject twice daily.
  • a unit dose can be about one-fourth of the daily amount with two unit doses administered to the subject twice daily.
  • a unit dose comprises about, less than about, or more than about 0.05 mg SNP or 100 mg L-arginine. In some embodiments, a unit dose comprises about, less than about, or more than about 50 mg resveratrol. In some embodiments, a unit dose comprises about, less than about, or more than about 50 mg metformin. In some embodiments, a unit dose comprises about, less than about, or more than about 550 mg leucine. In some embodiments, a unit dose comprises about, less than about, or more than about 200 mg of one or more leucine metabolites. In some embodiments, a unit dose (e.g. a unit dose comprising leucine) is administered as two unit doses two times per day. In some embodiments, a unit dose (e.g. a unit dose comprising one or more leucine metabolites, such as HMB) is administered as one unit dose two timer per day.
  • a unit dose e.g. a unit dose comprising leucine metabolites, such as H
  • the agents described herein can be administered to a subject orally or by any other methods.
  • Methods of oral administration include administering the composition as a liquid, a solid, or a semi- solid that can be taken in the form of a dietary supplement or a food stuff.
  • the agents described herein can be co- administered.
  • the agents can be administered simultaneously, e.g., in a single composition, or can be administered sequentially.
  • the agents can be administered sequentially within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 30, 60, 90, or 120 minutes from each other.
  • the agents described herein can be administered periodically.
  • the agents can be administered one, two, three, four times a day, or even more frequent.
  • the subject can be administered every 1, 2, 3, 4, 5, 6 or 7 days.
  • the agents are administered three times daily.
  • the administration can be concurrent with meal time of a subject.
  • the period of treatment or diet supplementation can be for about 1, 2, 3, 4, 5, 6, 7, 8, or 9 days, 2 weeks, 1- 11 months, or 1 year, 2 years, 5 years or even longer.
  • the subject is administered the agents for six weeks or more.
  • the dosages that are administered to a subject can change or remain constant over the period of treatment.
  • the daily dosing amounts can increase or decrease over the period of administration.
  • the length of the period of administration and/or the dosing amounts can be determined by a physician, or any other type of clinician.
  • the physician or clinician can observe the subject's response to the administered compositions and adjust the dosing based on the subject's performance. For example, dosing for subjects that show reduced effects in energy regulation can be increased to achieve desired results.
  • compositions for use in practicing any of the methods can comprise any combination of the agents described herein.
  • a composition can comprise one, two, three, four, or more than four of the agents described herein.
  • compositions described herein can be compounded into a variety of different dosage forms.
  • compositions can be formulated for oral administration, e.g., as a tablet, chewable tablet, caplets, capsule, soft gelatin capsules, lozenges or solution.
  • Compositions can be formulated as a nasal spray or for injection when in its solution form.
  • the composition is a liquid composition suitable for oral consumption.
  • compositions suitable for oral administration can be presented as discrete dosage forms, such as capsules, cachets, or tablets, or liquids or aerosol sprays each containing a predetermined amount of an active ingredient as a powder or in granules, a solution, or a suspension in an aqueous or non-aqueous liquid, an oil-in-water emulsion, or a water-in-oil liquid emulsion, including liquid dosage forms (e.g., a suspension or slurry), and oral solid dosage forms (e.g. , a tablet or bulk powder).
  • liquid dosage forms e.g., a suspension or slurry
  • oral solid dosage forms e.g. , a tablet or bulk powder
  • Oral dosage forms may be formulated as tablets, pills, dragees, capsules, emulsions, lipophilic and hydrophilic suspensions, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by an individual or a patient to be treated.
  • Such dosage forms can be prepared by any of the methods of formulation.
  • the active ingredients can be brought into association with a carrier, which constitutes one or more necessary ingredients.
  • Capsules suitable for oral administration include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • filler such as lactose
  • binders such as starches
  • lubricants such as talc or magnesium stearate
  • the composition for oral use can be obtained by mixing a composition a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP).
  • the compositions are prepared by uniformly and intimately admixing the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation.
  • a tablet can be prepared by compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets can be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as powder or granules, optionally mixed with an excipient such as, but not limited to, a binder, a lubricant, an inert diluent, and/or a surface active or dispersing agent. Molded tablets can be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
  • compositions may be in liquid form.
  • Exemplary liquid forms which may be formulated for oral administration or for administration by injection, include aqueous solution, suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil, or peanut oil as well as elixirs and similar pharmaceutical vehicles.
  • Suitable dispersing or suspending agents for aqueous suspensions include synthetic natural gums, such as tragacanth, acacia, alginate, dextran, sodium
  • a method may comprise administering to a subject a combination of an injectable composition and a composition for oral administration.
  • Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for reconstitution with water or other suitable vehicles before use.
  • Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, methyl cellulose or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters or ethyl alcohol); preservatives (e.g., methyl or propyl p-hydroxybenzoates or sorbic acid); and artificial or natural colors and/or sweeteners.
  • suspending agents e.g., sorbitol syrup, methyl cellulose or hydrogenated edible fats
  • emulsifying agents e.g., lecithin or acacia
  • non-aqueous vehicles e.g., almond oil, oily esters or ethyl alcohol
  • preservatives e.g., methyl or propyl p-hydroxybenzoates or sorbic acid
  • the preparation of pharmaceutical compositions can be conducted in accordance with generally accepted procedures for the preparation of pharmaceutical preparations. See, for example, Remington's Pharmaceutical Sciences 18th Edition (1990), E. W. Martin ed., Mack Publishing Co., PA. Depending on the intended use and mode of administration, it may be desirable to process the magnesium-counter ion compound further in the preparation of pharmaceutical compositions. Appropriate processing may include mixing with appropriate nontoxic and non-interfering components, sterilizing, dividing into dose units, and enclosing in a delivery device.
  • Certain embodiments further encompass anhydrous compositions and dosage forms comprising an active ingredient, since water can facilitate the degradation of some compounds.
  • water may be added (e.g., 5%) in the arts as a means of simulating long-term storage in order to determine characteristics such as shelf-life or the stability of formulations over time.
  • Anhydrous compositions and dosage forms can be prepared using anhydrous or low moisture containing ingredients and low moisture or low humidity conditions. Compositions and dosage forms which contain lactose can be made anhydrous if substantial contact with moisture and/or humidity during manufacturing, packaging, and/or storage is expected. An anhydrous composition may be prepared and stored such that its anhydrous nature is maintained.
  • anhydrous compositions may be packaged using materials known to prevent exposure to water such that they can be included in suitable formulary kits.
  • suitable packaging include, but are not limited to, hermetically sealed foils, plastic or the like, unit dose containers, blister packs, and strip packs.
  • An agent described herein can be combined in an intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques.
  • the carrier can take a wide variety of forms depending on the form of preparation desired for administration.
  • any of the usual pharmaceutical media can be employed as carriers, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, and the like in the case of oral liquid preparations (such as suspensions, solutions, and elixirs) or aerosols; or carriers such as starches, sugars, micro-crystalline cellulose, diluents, granulating agents, lubricants, binders, and disintegrating agents can be used in the case of oral solid preparations, in some embodiments without employing the use of lactose.
  • suitable carriers include powders, capsules, and tablets, with the solid oral preparations. If desired, tablets can be coated by standard aqueous or nonaqueous techniques.
  • Some examples of materials which may serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum
  • Binders suitable for use in dosage forms include, but are not limited to, corn starch, potato starch, or other starches, gelatin, natural and synthetic gums such as acacia, sodium alginate, alginic acid, other alginates, powdered tragacanth, guar gum, cellulose and its derivatives (e.g., ethyl cellulose, cellulose acetate, carboxymethyl cellulose calcium, sodium carboxymethyl cellulose), polyvinyl pyrrolidone, methyl cellulose, pre-gelatinized starch, hydro xypropyl methyl cellulose, microcrystalline cellulose, and mixtures thereof.
  • natural and synthetic gums such as acacia, sodium alginate, alginic acid, other alginates, powdered tragacanth, guar gum, cellulose and its derivatives (e.g., ethyl cellulose, cellulose acetate, carboxymethyl cellulose calcium, sodium carboxymethyl cellulose), polyvinyl pyrrolidon
  • Lubricants which can be used to form compositions and dosage forms include, but are not limited to, calcium stearate, magnesium stearate, mineral oil, light mineral oil, glycerin, sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid, sodium lauryl sulfate, talc, hydrogenated vegetable oil (e.g., peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil), zinc stearate, ethyl oleate, ethylaureate, agar, or mixtures thereof.
  • Additional lubricants include, for example, a syloid silica gel, a coagulated aerosol of synthetic silica, or mixtures thereof.
  • a lubricant can optionally be added, in an amount of less than about 1 weight percent of the composition.
  • Lubricants can be also be used in conjunction with tissue barriers which include, but are not limited to, polysaccharides, polyglycans, seprafilm, interceed and hyaluronic acid.
  • Disintegrants may be used in the compositions to provide tablets that disintegrate when exposed to an aqueous environment. Too much of a disintegrant may produce tablets which may disintegrate in the bottle. Too little may be insufficient for disintegration to occur and may thus alter the rate and extent of release of the active ingredient(s) from the dosage form. Thus, a sufficient amount of disintegrant that is neither too little nor too much to detrimentally alter the release of the active ingredient(s) may be used to form the dosage forms of the compounds disclosed herein.
  • Disintegrants that can be used to form compositions and dosage forms include, but are not limited to, agar-agar, alginic acid, calcium carbonate, microcrystalline cellulose, croscarmellose sodium, crospovidone, polacrilin potassium, sodium starch glycolate, potato or tapioca starch, other starches, pre- gelatinized starch, other starches, clays, other algins, other celluloses, gums or mixtures thereof.
  • suitable fillers for use in the compositions and dosage forms disclosed herein include, but are not limited to, talc, calcium carbonate (e.g., granules or powder), microcrystalline cellulose, powdered cellulose, dextrates, kaolin, mannitol, silicic acid, sorbitol, starch, pre-gelatinized starch, and mixtures thereof.
  • the active ingredient therein may be combined with various sweetening or flavoring agents, coloring matter or dyes and, if so desired, emulsifying and/or suspending agents, together with such diluents as water, ethanol, propylene glycol, glycerin and various combinations thereof.
  • the tablets can be uncoated or coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.
  • a time delay material such as glyceryl monostearate or glyceryl distearate can be employed.
  • Formulations for oral use can also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example, peanut oil, liquid paraffin or olive oil.
  • compositions may include a solubilizer to ensure good solubilization and/or dissolution of the agent and to minimize precipitation of the agent. This can be useful for compositions for non-oral use, e.g., compositions for injection.
  • a solubilizer may also be added to increase the solubility of the hydrophilic drug and/or other components, such as surfactants, or to maintain the composition as a stable or homogeneous solution or dispersion.
  • the composition can further include one or more pharmaceutically acceptable additives and excipients.
  • additives and excipients include, without limitation, detackifiers, anti-foaming agents, buffering agents, polymers, antioxidants, preservatives, chelating agents, viscomodulators, tonicifiers, flavorants, colorants, odorants, opacifiers, suspending agents, binders, fillers, plasticizers, lubricants, and mixtures thereof.
  • excipients includes monoglycerides, magnesium stearate, modified food starch, gelatin, microcrystalline cellulose, glycerin, stearic acid, silica, yellow beeswax, lecithin,
  • hydroxypropylcellulose croscarmellose sodium, and crospovidone.
  • compositions described herein can also be formulated as extended-release, sustained-release or time-release such that one or more components are released over time.
  • Delayed release can be achieved by formulating the one or more components in a matrix of a variety of materials or by microencapsulation.
  • the compositions can be formulated to release one or more components over a time period of 4, 6, 8, 12, 16, 20, or 24 hours.
  • the release of the one or more components can be at a constant or changing rate.
  • the one or more cofactors can be released in its dosage form at a slower rate than observed for an immediate release formulation of the same quantity of components.
  • the rate of change in the biological sample measured as the change in concentration over a defined time period from administration to maximum concentration for an controlled release formulation is less than about 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% of the rate of the immediate release formulation.
  • the rate of change in concentration over time is less than about 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% of the rate for the immediate release formulation.
  • the rate of change of concentration over time is reduced by increasing the time to maximum concentration in a relatively proportional manner. For example, a two-fold increase in the time to maximum concentration may reduce the rate of change in concentration by approximately a factor of 2. As a result, the one or more cofactors may be provided so that it reaches its maximum concentration at a rate that is significantly reduced over an immediate release dosage form.
  • the compositions may be formulated to provide a shift in maximum concentration by 24 hours, 16 hours, 8 hours, 4 hours, 2 hours, or at least 1 hour.
  • the associated reduction in rate of change in concentration may be by a factor of about 0.05, 0.10, 0.25, 0.5 or at least 0.8.
  • this is accomplished by releasing less than about 30%, 50%, 75%, 90%, or 95% of the one or more cofactors into the circulation within one hour of such administration.
  • the controlled release formulations exhibit plasma concentration curves having initial (e.g., from 2 hours after administration to 4 hours after administration) slopes less than 75%, 50%, 40%, 30%, 20% or 10% of those for an immediate release formulation of the same dosage of the same cofactor.
  • the rate of release of the cofactor as measured in dissolution studies is less than about 80%, 70%, 60% 50%, 40%, 30%, 20%, or 10% of the rate for an immediate release formulation of the same cofactor over the first 1, 2, 4, 6, 8, 10, or 12 hours.
  • the controlled release formulations provided herein can adopt a variety of formats.
  • the formulation is in an oral dosage form, including liquid dosage forms (e.g., a suspension or slurry), and oral solid dosage forms (e.g., a tablet or bulk powder), such as, but not limited to those, those described herein.
  • the controlled release tablet of a formulation disclosed herein can be of a matrix, reservoir or osmotic system. Although any of the three systems is suitable, the latter two systems can have more optimal capacity for encapsulating a relatively large mass, such as for the inclusion of a large amount of a single cofactor, or for inclusion of a plurality of cofactors, depending on the genetic makeup of the individual.
  • the slow-release tablet is based on a reservoir system, wherein the core containing the one or more cofactors is encapsulated by a porous membrane coating which, upon hydration, permits the one or more cofactors to diffuse through. Because the combined mass of the effective ingredients is generally in gram quantity, an efficient delivery system can provide optimal results.
  • tablets or pills can also be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action.
  • the tablet or pill can comprise an inner dosage an outer dosage component, the latter being in the form of an envelope over the former.
  • the two components can be separated by an enteric layer which serves to resist disintegration in the stomach and permits the inner component to pass intact into the duodenum or to be delayed in release.
  • enteric layers or coatings such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol and cellulose acetate.
  • a formulation comprising a plurality of cofactors may have different cofactors released at different rates or at different times. For example, there can be additional layers of cofactors interspersed with enteric layers.
  • Methods of making sustained release tablets are known in the art, e.g., see U.S. Patent Publications 2006/051416 and 2007/0065512, or other references disclosed herein.
  • Methods such as described in U.S. Patent Nos. 4,606,909, 4,769,027, 4,897,268, and 5,395,626 can be used to prepare sustained release formulations of the one or more cofactors determined by the genetic makeup of an individual.
  • the formulation is prepared using OROS® technology, such as described in U.S. Patent Nos. 6,919,373, 6,923,800, 6,929,803, and 6,939,556.
  • Other methods such as described in U.S. Patent Nos. 6,797,283, 6,764,697, and 6,635,268, can also be used to prepare the formulations disclosed herein.
  • the compositions can be formulated in a food composition.
  • the compositions can be a beverage or other liquids, solid food, semi-solid food, with or without a food carrier.
  • the compositions can include a black tea supplemented with any of the compositions described herein.
  • the composition can be a dairy product supplemented any of the compositions described herein.
  • the compositions can be formulated in a food composition.
  • the compositions can comprise a beverage, solid food, semi-solid food, or a food carrier.
  • liquid food carriers such as in the form of beverages, such as supplemented juices, coffees, teas, sodas, flavored waters, and the like can be used.
  • the beverage can comprise the formulation as well as a liquid component, such as various deodorant or natural carbohydrates present in conventional beverages.
  • natural carbohydrates include, but are not limited to, monosaccharides such as, glucose and fructose; disaccharides such as maltose and sucrose; conventional sugars, such as dextrin and cyclodextrin; and sugar alcohols, such as xylitol and erythritol.
  • Natural deodorant such as taumatin, stevia extract, levaudioside A, glycyrrhizin, and synthetic deodorant such as saccharin and aspartame may also be used.
  • Agents such as flavoring agents, coloring agents, and others can also be used.
  • pectic acid and the salt thereof, alginic acid and the salt thereof, organic acid, protective colloidal adhesive, pH controlling agent, stabilizer, a preservative, glycerin, alcohol, or carbonizing agents can also be used.
  • Fruit and vegetables can also be used in preparing foods or beverages comprising the formulations discussed herein.
  • compositions can be a snack bar supplemented with any of the compositions described herein.
  • the snack bar can be a chocolate bar, a granola bar, or a trail mix bar.
  • the present dietary supplement or food can be a chocolate bar, a granola bar, or a trail mix bar.
  • compositions are formulated to have suitable and desirable taste, texture, and viscosity for consumption.
  • Any suitable food carrier can be used in the present food compositions.
  • Food carriers include practically any food product. Examples of such food carriers include, but are not limited to food bars (granola bars, protein bars, candy bars, etc.), cereal products (oatmeal, breakfast cereals, granola, etc.), bakery products (bread, donuts, crackers, bagels, pastries, cakes, etc.), beverages (milk-based beverage, sports drinks, fruit juices, alcoholic beverages, bottled waters), pastas, grains (rice, corn, oats, rye, wheat, flour, etc.), egg products, snacks (candy, chips, gum, chocolate, etc.), meats, fruits, and vegetables.
  • food carriers employed herein can mask the undesirable taste (e.g., bitterness).
  • the food composition presented herein exhibit more desirable textures and aromas than that of any of the components described herein.
  • liquid food carriers may be used to obtain the present food compositions in the form of beverages, such as supplemented juices, coffees, teas, and the like.
  • solid food carriers may be used to obtain the present food
  • compositions in the form of meal replacements such as supplemented snack bars, pasta, breads, and the like.
  • semi-solid food carriers may be used to obtain the present food compositions in the form of gums, chewy candies or snacks, and the like.
  • the dosing of the combination compositions can be administered about, less than about, or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times a daily.
  • a subject can receive dosing for a period of about, less than about, or greater than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more days, weeks or months.
  • a unit dose can be a fraction of the daily dose, such as the daily dose divided by the number of unit doses to be administered per day.
  • a unit dose can be a fraction of the daily dose that is the daily dose divided by the number of unit doses to be administered per day and further divided by the number of unit doses (e.g. tablets) per administration.
  • the number of unit doses per administration may be about, less than about, or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.
  • the number of doses per day may be about, less than about, or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.
  • the number of unit doses per day may be determined by dividing the daily dose by the unit dose, and may be about, less than about, or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, 20, or more unit doses per day.
  • a unit dose can be about 1/2, 1/3, 1/4, 1/5, 1/6, 1/7, 1/8, 1/9, 1/10.
  • a unit dose can be about one-third of the daily amount and administered to the subject three times daily.
  • a unit dose can be about one-half of the daily amount and administered to the subject twice daily.
  • a unit dose can be about one-fourth of the daily amount with two unit doses administered to the subject twice daily.
  • a unit dose comprises about, less than about, or more than about 50 mg resveratrol.
  • a unit dose comprises about, less than about, or more than about 550 mg leucine.
  • a unit dose comprises about, less than about, or more than about 200 mg of one or more leucine metabolites.
  • a unit dose e.g. a unit dose comprising leucine
  • a unit dose is administered as two unit doses two times per day.
  • a unit dose e.g. a unit dose comprising one or more leucine metabolites, such as HMB
  • Compositions disclosed herein can further comprise a flavorant and can be a solid, liquid, gel or emulsion.
  • a composition comprising a unit dosage form can be formulated to provide a dosage of an NO donor and a branched chain amino acid in free amino acid form, or a metabolite thereof.
  • the dosage of the NO donor can be an amount of about 0.05 mg, about 0.1 mg, about 0.2 mg, about 0.3 mg, about 0.4 mg, about 0.5 mg, about 0.6 mg, about 0.7 mg, about 0.8 mg, about 0.9 mg, about 1 mg, about 1.1 mg, about 1.2 mg, about 1.3, about 1.4 mg, about 1.5 mg, about 1.6 mg, about 1.7 mg, about 1.8 mg, about 1.9 mg, about 2 mg, about 2.1 mg, about 2.5 mg, about 3 mg, about 4 mg, about 5 mg, about 10 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 150 mg, about 160 mg, about 180 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg
  • An sub-therapeutic dosage of the NO donor can be an amount of about 0.01 mg, about 0.02 mg, about 0.03 mg, about 0.04 mg, about 0.05 mg, about 0.06 mg, about 0.07 mg, about 0.08 mg, about 0.09 mg, about 0.1 mg, about 0.2 mg, about 0.3 mg, about 0.4 mg, about 0.5 mg, about 0.6 mg, about 0.7 mg, about 0.8 mg, about 0.9 mg, about 1 mg, about 1.1 mg, about 1.2 mg, about 1.3, about 1.4 mg, about 1.5 mg, about 1.6 mg, about 1.7 mg, about 1.8 mg, about 1.9 mg, about 2 mg, about 2.1 mg, about 2.5 mg, about 3 mg, about 4 mg, about 5 mg, about 10 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 150 mg, about 160 mg, about 180 mg, about 200 mg or any intermediate ranges or values.
  • the dosage of the branched chain amino acid in free amino acid form or metabolite thereof can be a therapeutic dose or sub-therapeutic dose.
  • composition can comprise about 10-3000 mg (e.g., 50, 100, 200, 250, 300, 400, 500, 750, 1000, 1125, 1500, or 3000 mg) of leucine in free amino acid form, 5-500 mg of HMB (e.g., 5, 10, 20, 50, 100, 200, 300, 400, or 500 mg of HMB), about 20 to about 300 mg of KIC (e.g., 20, 50, 100, 200, 300 mg of KIC), or any combination thereof.
  • any of the above unit dosage compositions can further comprise an additional agent.
  • the additional agent can be a sirtuin pathway activator and/or PDE inhibitor. Exemplary sirtuin pathway activators and PDE inhibitors are described herein.
  • a weight % of the additional agent in the composition, excluding excipients (e.g., fillers) is 5-50%.
  • the additional agent is metformin. The metformin can be present in the unit dosage composition in a therapeutic or sub-therapeutic dose.
  • the unit dosage composition can further comprise about 2.5 to about 500 mg of metformin (e.g., about 2.5, 5, 10, 20, 25, 50, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, or 500 mg of metformin).
  • the additional agent is a PDE inhibitor (e.g., a PDE5 inhibitor).
  • the additional agent is icariin.
  • the icariin can be present in the unit dosage composition in a therapeutic or sub-therapeutic dose.
  • the unit dosage composition can further comprise about 1 to about 2000 mg of icariin (e.g., about 1, 10, 25, 50, 100, 500, 600, 700, 800, 900, 1000, 1500, or 2000 mg of icariin).
  • the additional agent is sildenafil.
  • the sildenafil can be present in the unit dosage composition in a therapeutic or subtherapeutic dose.
  • the unit dosage composition can further comprise about 1 to about 2000 mg of sildenafil (e.g., about 1, 10, 25, 50, 100, 500, 600, 700, 800, 900, 1000, 1500, or 2000 mg of sildenafil).
  • the unit dosage can comprise about 250 to about 2000 mg of leucine.
  • the unit dosage can comprise about 500 to about 1500 mg of leucine.
  • the unit dosage can comprise about 900 to about 1300 mg of leucine.
  • the unit dosage can comprise about 0.01 to about 100 mg of a NO donor.
  • the weight ratio of NO donor relative to leucine can be 0.00002-0.2.
  • the weight ratio of NO donor relative to leucine can be 0.00002-0.0001.
  • the weight ratio of NO donor relative to leucine can be 0.01-0.2.
  • the unit dosage can further comprise metformin.
  • the weight ratio of metformin relative to leucine can be 0.01-0.6.
  • the weight ratio of metformin relative to leucine can be 0.1-0.5.
  • the weight ratio of metformin relative to leucine can be 0.15- 0.3.
  • the unit dosage can optionally further comprise resveratrol.
  • An exemplary unit dosage composition comprises about 125 mg metformin, 1.125 g leucine and, optionally, 50 mg resveratrol.
  • a unit dosage can comprise about 50 mg metformin, 1.125 g leucine and optionally 50 mg resveratrol.
  • a unit dosage can comprise about 250 mg metformin, 1.125 g leucine and optionally 50 mg resveratrol.
  • a unit dosage can comprise about 500 mg metformin, 1.125 g leucine and optionally 50 mg resveratrol.
  • An exemplary formulation of a unit dosage comprising leucine and an NO donor e.g., L-Arginine
  • An exemplary formulation of a unit dosage comprising leucine and an NO donor (e.g.,
  • compositions with with (a) branched chain amino acids and/or metabolites thereof, such as leucine or leucine metabolites, e.g. , HMB or KIC; and (b) one or more NO donors.
  • the molar ratio of (a) branched chain amino acids and/or metabolites thereof to (b) one or more NO donors about or greater than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 120, or 150 or any intermediate ranges or numbers.
  • the molar ratio of one or more branched chain amino acids and/or metabolites thereof to one or more NO donors contained in the subject compositions is about or greater than about 20, 30, 40, 50, 60, 70, 80, 90, 95, 90, 95, 100, 105, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 250, 300, 350, 400, or 500.
  • the molar ratio of component (a) to (b) in said composition is greater than about 20, 40, 60, 80, 100, 120, or 150.
  • the molar ratio of component (a) to (b) in said composition is greater than about 80, 100, 120, or 150.
  • the molar ratio of component (a) to (b) in said composition is greater than about 80, 100, 120, or 150. In some embodiments, the molar ratio of component (a) to (b) in said composition is greater than about 200, 250, or 300. In some embodiments, the molar ratio of component (a) to (b) in said composition is greater than about 40, 150, 250, or 500.
  • the dosing of NO donor, leucine, any metabolites of leucine, the PDE inhibitor can be designed to achieve a specified physiological concentration or circulating level of leucine, metabolites of leucine and/or a PDE 5 inhibitor.
  • the physiological concentration can be a circulating level as measured in the blood stream of a subject.
  • the subject can be a human or an animal.
  • a selected dosing can be altered based on the characteristics of the subject, such as weight, rate of energy metabolism, genetics, ethnicity, height, or any other characteristic.
  • the amount of leucine in a unit dose can be such that the circulating level of leucine in a subject is about or greater than about 0.25 mM, 0.5 mM, 0.75 mM, or 1 mM.
  • a dosing of about 1,125 mg leucine (e.g., free leucine) can achieve a circulating level of leucine in a subject that is about 0.5 mM.
  • a dosing of about 300 mg leucine (e.g., free leucine) can achieve a circulating level of leucine in a subject that is about 0.25 mM.
  • the dosing of about sildenafil can achieve a circulating concentration of about or less than about 0.1, 0.5, 1, 2, 5, or 10 nM.
  • the target or achieved circulating concentration of sildenafil is less than about 1 nM.
  • a unit dose of about 20 mg of sildenafil can achieve a circulating concentration of about 100 nM of sildenafil.
  • a unit dose of about 0.2 mg of sildenafil can achieve a circulating concentration of about 1 nM of sildenafil.
  • the dosing of about icariin can achieve a circulating concentration of about or less than about 0.1, 0.5, 1, 2, 5, or 10 nM.
  • the target or achieved circulating concentration of icariin is less than about 1 nM.
  • a unit dose of about 20 mg of icariin can achieve a circulating concentration of about 100 nM of icariin.
  • a unit dose of about 0.1 mg of icariin can achieve a circulating concentration of about 1 nM of icariin.
  • the molar or mass ratios are circulating molar or mass ratios achieved after administration one or more compositions to a subject.
  • the compositions can be a combination composition described herein.
  • the molar ratio of a combination composition in a dosing form can be adjusted to achieve a desired circulating molar ratio.
  • the molar ratio can be adjusted to account for the bioavailability, the uptake, and the metabolic processing of the one or more components of a combination composition. For example, if the bioavailability of a component is low, then the molar amount of a that component can be increased relative to other components in the combination composition.
  • the circulating molar or mass ratio is achieved within about 0.1, 0.5, 0.75, 1, 3, 5, or 10, 12, 24, or 48 hours after administration.
  • the circulating molar or mass ratio can be maintained for a time period of about or greater than about 0.1, 1, 2, 5, 10, 12, 18, 24, 36, 48, 72, or 96 hours.
  • the circulating molar ratio of leucine to sildenafil is about or greater than about 100,000, 250,000, 500,000, 750,000 or more. In some embodiments, the circulating molar ratio of HMB to sildenafil is about or greater than about 1,000, 2,500, 5,000, 7,500 or more. In some embodiments, the circulating molar ratio of resveratrol to sildenafil is about or greater than about 50, 100, 200, 400, 800 or more.
  • compositions can be administered to a subject such that the subject is
  • the total daily dose can be determined by the sum of doses administered over a 24 hour period.
  • the total daily dose of the composition can include about 0.05, 0.1, 0.5, 1, 2, 5, 10, 20, 40, 60, 80, or 100 mg of sildenafil.
  • the total daily dose of the composition can include about 1, 10, 20, 50, 100, 150, 300, 400, 500, 750, 1000, 1500, or 2000 mg of icariin.
  • the total daily dose of the composition can include about 1, 10, 20, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 900, or 1000 mg of metformin.
  • the total daily dose of the composition can include at least about 250, 500, 750, 1000, 1125, 2000, 2250 mg, 2550 mg or more of a branched chain amino acid or metabolite thereof.
  • the branched chain amino acid can be leucine, HMB, or any other branched chain amino acid described herein.
  • a selected dose of a composition can be administered to a subject such that the subject achieves a desired circulating level of the composition.
  • the desired circulating level of the leucine can be at least about 0.25, 0.5, 0.75, 1 mM or more of leucine.
  • the desired circulating level of the sildenafil can be about 0.1, 0.5, 1, 2, 5, 10 nM or more of sildenafil.
  • the desired circulating level of the icariin can be about 0.1, 0.5, 1, 2, 5, 10 nM or more of icariin.
  • the desired circulating level of the metformin can be about 1, 2, 4, 5 ⁇ or more of metformin.
  • the desired circulating level of the rosiglitazone can be about 1, 10, 25, 50, 100, 400 nM or more of rosiglitazone.
  • the desired circulating level of the pioglitazone can be about 0.25, 0.50, 1.0, 2.0 ⁇ or more of pioglitazone.
  • the selected dose can be chosen based on the characteristics of the subject, such as weight, height, ethnicity, or genetics.
  • kits include one or more compositions described herein, particularly one or more NO donors and one or more branched amino acids, such as leucine and/or a leucine metabolite, in suitable packaging, and written material that can include instructions for use, discussion of clinical studies, listing of side effects, and the like.
  • kits may also include information, such as scientific literature references, package insert materials, clinical trial results, and/or summaries of these and the like, which indicate or establish the activities and/or advantages of the composition, and/or which describe dosing,
  • the kit may further contain another agent.
  • two or more agents are provided as separate compositions in separate containers within the kit.
  • two or more agents are provided as a single composition within a container in the kit.
  • Suitable packaging and additional articles for use e.g., measuring cup for liquid
  • Kits described herein can be provided, marketed and/or promoted to health providers, including physicians, nurses, pharmacists, formulary officials, and the like. Kits may also, in some embodiments, be marketed directly to the consumer.
  • a kit can comprise a multi-day supply of unit dosages.
  • the unit dosages can be any unit dosage described herein.
  • the kit can comprise instructions directing the administration of the multi-day supply of unit dosages over a period of multiple days.
  • the multi- day supply can be a one-month supply, a 30-day supply, or a multi-week supply.
  • the multi-day supply can be a 90-day, 180-day, 3-month or 6-month supply.
  • the kit can include packaged daily unit dosages, such as packages of 1, 2, 3, 4, or 5 unit dosages.
  • the kit can be packaged with other dietary supplements, vitamins, and meal replacement bars, mixes, and beverages.
  • a kit can further comprise a wearable activity monitor.
  • the wearable activity monitor can monitor physical and/or ambulatory activity via a pedometer, accelerometer, or Exemplary wearable activity monitors include, e.g.; a Fitbit®, Jawbone UP®, LarkLifeTM, Nike FuelBand, Striiv Play, BodyMedia FitCore, among others.
  • Such wearable activity monitors are described in US Patent Nos: 8,403,845; 8,398,546; 8,382,590; 8,369,936; 8,275,635; 8,157,731; 8,073,707; 7,959,567; 7,689,437; 7,502,643; 7,285,090; 7,261,690;
  • kits can further comprise instructions for the subject to engage in a physical activity regimen in addition to instructions for use of the composition.
  • Such kits can further comprise instructions for use of the wearable activity monitors.
  • Such kits can also comprise wearable activity monitor accessories, such as, e.g., a charger, and/or a port or wireless for communicating data from the activity monitor to a computer or server.
  • Example 1 Interactive effect of Leucine and NO donors as a therapeutic for
  • SNP/metformin SNP/metformin or individual components are assessed on Sirtl/AMPK activation, fatty acid oxidation (Seahorse), and lipid accumulation (OilRed staining), and the attenuation of the LPS- induced inflammatory response.
  • L-Arginine or SNP with leucine and rosiglitazone are assessed on Sirtl/AMPK activation, fatty acid oxidation (Seahorse), and lipid accumulation (OilRed staining), and the attenuation of the LPS- induced inflammatory response.
  • L-Arginine or SNP with metformin, leucine, rosiglitazone, and sildenafil as compared to two-way combinations (L-Arginine/Leucine, L-Arginine/metformin, SNP/Leucine, SNP/metformin, L-Arginine/rosiglitazone, SNP/ rosiglitazone), three-way combinations (L-Arginine/Leucine/metformin, L-Arginine/Leucine/rosiglitazone, L- Arginine/rosiglitazone/ metformin, SNP/Leucine/metformin, SNP/Leucine/rosiglitazone, SNP/rosiglitazone/metformin), four way combinations (L-
  • Arginine/Leucine/metformin/sildenafil L-Arginine/Leucine/rosiglitazone/sildenafil, L- Arginine/rosiglitazone/metformin/sildenafil, SNP/Leucine/metformin/sildenafil,
  • SNP/Leucine/rosiglitazone/sildenafil, SNP/rosiglitazone/metformin/sildenafil) or individual components are assessed on Sirtl/AMPK activation, fatty acid oxidation (Seahorse), and lipid accumulation (OilRed staining), and the attenuation of the LPS-induced inflammatory response.
  • Human HepG2 liver cells (ATCC ® , HB-8065TM) were grown and maintained in low glucose (5 mM) DMEM containing 10% fetal bovine serum (FBS) and antibiotics (1% penicillin- streptomycin) at 37°C in 5% C0 2 according to manufacturer's protocol. For all experiments, cells were seeded in low glucose medium. The following day, the medium was changed to high glucose (25 mM) DMEM containing 100 nM insulin, 10% FBS and antibiotics for 48 hours to induce lipid accumulation (except for low glucose control cells). The last 12 to 16 hours, the FBS content was changed to 0.2% FBS. After the 48 hours, the treatment was added to the high glucose media for the indicated time.
  • low glucose 5 mM
  • FBS fetal bovine serum
  • antibiotics 1% penicillin- streptomycin
  • Mouse RAW264.7 is grown and maintained in high glucose DMEM with 10% FBS. For experiments, 24 hours after seeding cells are activated with LPS (10 nmg/ml) for an additional 24 hours before treatment.
  • HAVEC Human umbilical vein endothelial cells
  • ATCC® PCS- 100-010TM
  • Vascular Cell Basal Medium ATCC PCS- 100-030
  • ATCC PCS- 100-030 containing the Endothelial Cell Growth Kit-BBE (ATCC PCS- 100-040, containing Bovine Brain Extract, rh EGF, L-glutamine, heparin sulfate, hydrocortisone hemisuccinate, Fetal Bovine Serum and ascorbic acid) and antibiotics (1% penicillin- streptomycin) at 37°C in 5% C0 2 according to manufacturer's protocol.
  • antibiotics 1% penicillin- streptomycin
  • TNFcc and MCP1 are assessed via the ELISA kits from Abeam (abl00747-TNF alpha Mouse ELISA Kit and abl00721_MCPl Mouse LISA Kit) according to manufacturer's instructions.
  • Sirtl activity is measured with the Sirtl FRET-based screening assay kit (Cayman Chemical Company, Ann Arbor, MI, USA). Cell extracts of treated HepG2 cells (5 ⁇ > are incubated with Sirtl substrate under low NAD+ concentration (50 ⁇ ) for 30 minutes according to manufacturer's protocol. The fluorescence is measured with excitation and emission wavelengths of 360 nm and 450 nm, respectively. The increase in fluorescence is proportional to the amount of deacetylated substrate and thus Sirtl activity.
  • Nitric oxide (NO) content Treated HepG2 cells were washed twice with PBS, then incubated with DAF-FM diacetate (5 uM final working concentration) diluted in phenol-red free DMEM for 30 minutes at 37°C. Then cells were washed three times with PBS and cells were incubated for an additional 20 minutes at 37°C to allow complete de-esterification of the intracellular diacetates. NO was detected by fluorescence excitation and emission maxima of 495 and 515 nm, respectively.
  • Example 2 Interactive effects of leucine-metformin-NQ donor versus leucine- metformin and leucine-NQ donor in human of non-alcoholic steatohepatitis (NASH).
  • NASH non-alcoholic steatohepatitis
  • Metformin 250 mg, administered twice a day
  • Leucine 1.1 g, administered twice a day
  • sildenafil (1 mg, administered twice a day) + metformin (250 mg, administered twice a day)
  • ginine 500 mg, administered twice a day
  • Leucine 1.1 g, administered twice a day
  • L-arginine 500 mg, administered twice a day
  • Leucine (1.1 g, administered twice a day) + SNP (0.05 mg, administered twice a day)
  • L- Leucine (1.1 g, administered twice a day) + L-arginine (500 mg, administered twice a day) + sildenafil (1 mg, administered twice a day) + metformin (250 mg, administered twice a day)
  • L- Leucine (1.1 g, administered twice a day) + SNP (0.05 mg, administered twice a day) + sildenafil (1 mg, administered twice a day) + metformin (250 mg, administered twice a day)
  • liver biopsy The effects of the treatments and diet on hepatic lipid accumulation are assessed using a liver biopsy, a blood test, a FibroTest, a SteatoTest, a gene expression profiling of the genes previously provided, or combinations thereof.
  • a liver biopsy is performed and improvement in
  • NAFLD Activity Score (NAS) is evaluated. An improvement of resolution of NASH is also evaluated.
  • the placebo group is expected to maintain significant hepatocellular injury. In response to the treatments, it is expected to improve NAFLD Activity Score by at least two points and to improve resolution of NASH in fibrosis in (a) the leucine and NO Donor (arginine or SNP) double combination, (b) the leucine, metformin and sildenafil three-way combination, and (c) the leucine, NO Donor, metformin and sildenafil four-way combination.
  • Example 3 Interactive effects of leucine and NO donor combination treatment on non-alcoholic fatty liver disease (NAFLD).
  • NAFLD non-alcoholic fatty liver disease
  • the combination treatment is a formulation of the essential amino acid L-leucine and NO donors (L-arginine or SNP). All unit doses are formulated in tablets or capsules. Each unit dose can comprise 1 tablet or 1 capsule, 2 tablets or 2 capsules, or 3 tablets or 3 capsules. In some cases, each tablet or capsule can comprise differing amounts of leucine and NO donors (L- arginine or SNP). In some cases, each tablet or capsule in a dose unit can be of different colors. For example, the various amount of leucine and NO donors (L-arginine or SNP) in a dose unit can be denoted by different colors.
  • these doses can be administered over 1 tablet or capsule, 2 tablets or capsules, or 3 tablets or capsules. All unit doses are provided in coded tablets or capsules to double-blind the medications to patients and trial staff.
  • the total daily dose of L-arginine is about 3 g, about 4 g, about 5 g, about 6 g, about 7 g, about 8 g, about 9 g, about 10 g, or less.
  • the total daily dose of SNP is about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 8 mg, about 10 mg, or less.
  • the total daily dose of L-leucine is about 1.1 g, about 2.2 g, or less.
  • Subjects meet the following criteria for inclusion in this study. Subjects are males or non-pregnant females, 18-75 years of age inclusive. Subjects have histological evidence of NASH based on diagnostic liver biopsy, either during screening or from historical liver biopsy within the preceding 90 days, and a NAS score >4, with a score of >1 in each NAS component (e.g., steatosis, ballooning, and lobular inflammation); have HbAlc of 9% or less, be otherwise stable health for preceding 12 weeks; and have BMI of 45 kg/m or less.
  • NAS component e.g., steatosis, ballooning, and lobular inflammation
  • subjects are not in the treatment or have been treated with NO donors such as oral nitrates, metformin, insulin, sildenafil or other PDE5 inhibitors such as tadalafil and vardenafil, alpha blockers, thiazoladinediones or medications associated with increased hepatic steatosis within the preceding four weeks.
  • NO donors such as oral nitrates, metformin, insulin, sildenafil or other PDE5 inhibitors such as tadalafil and vardenafil, alpha blockers, thiazoladinediones or medications associated with increased hepatic steatosis within the preceding four weeks.
  • Female subjects are not lactating, pregnant or unwilling to practice adequate contraception for duration of the study. Subjects do not have concomitant significant metabolic (other than diabetes), infectious, inflammatory, neoplastic or other non-liver disease.
  • alcohol abuse e.g., consume at least 7 drinks or more per week for females and at least 14 drinks or more per week for males
  • recreational drug use within the past 3 months or failure on urinary drug screen
  • currently or recent (within the previous four weeks) use of steroids or presence of concomitant disease requiring steroid use or recent (within the previous twelve weeks) serious infection, such as pneumonia or urinary tract infection.
  • NAFLD non-alcoholic fatty liver disease
  • Treatment efficacy is also evaluated by measuring liver fat by proton-density-fat-fraction (PDFF) magnetic resonance imaging (MRI) (MRI PDFF), evaluating serum alanine aminotransferase (ALT) level, evaluating serum gamma glutamyl transferase (GGT), evaluating hepatic necro-inflammation as measured by circulating cytokeratin 18 (K18) level, evaluating liver stiffness as measured by transient elastography (TE), evaluating hepatic steatosis as measured by TE, evaluating fasting glucose, insulin, HbAlc and insulin sensitivity (HOMA IR ), evaluating blood lipids (cholesterol, LDL particle fractions, HDL sub-fractions, Lipoprotein (a) "Lp(a)", triglycerides), and evaluating C-reactive protein (CRP) or inflammation.
  • PDFF proton-density-fat-fraction
  • MRI PDFF magnetic resonance imaging
  • ALT serum alan
  • Subjects receiving the combination treatment are expected to have histological improvement in NAFLD Activity Score (NAS) during the 12-month treatment, and changes in biological marker or biomarker levels over the 12-months period.
  • NAS NAFLD Activity Score
  • subjects respond to the combination treatment are expected to have reduced hepatic fat, reduced serum ALT level, reduced circulating K18 level, reduced liver fibrosis, reduced hepatic steatosis, reduced blood lipids, reduced inflammation, and increased insulin sensitivity.
  • the placebo group is expected to maintain significant hepatocellular injury, higher hepatic fat, higher blood lipids, higher inflammation, higher ALT level, and higher K18 level.
  • Example 4 Effects of leucine and NO donor combination treatment on non-alcoholic fatty liver disease (NAFLD) in children.
  • NAFLD non-alcoholic fatty liver disease
  • the combination treatment is a formulation of the essential amino acid L-leucine and NO donors (L-arginine or SNP). All unit doses are formulated in tablets or capsules. Each unit dose can comprise 1 tablet or 1 capsule, 2 tablets or 2 capsules, or 3 tablets or 3 capsules. In some cases, each tablet or capsule can comprise various amount of leucine and NO donors (L- arginine or SNP). In some cases, each tablet or capsule in a dose unit can be of different colors. For example, the various amount of leucine and NO donors (L-arginine or SNP) in a dose unit can be denoted by different colors.
  • these doses can be administered over 1 tablet or capsule, 2 tablets or capsules, or 3 tablets or capsules. All unit doses can be provided in coded tablets or capsules to double-blind the medications to patients and trial staff.
  • the total daily dose of L-leucine is about 1.1 g, about 2.2 g, or less.
  • the total daily dose of SNP is limited to be less than 10 mg.
  • the total daily dose of L-arginine is limited to be less than 10 g.
  • the total daily dose of sildenafil is limited to be less than 4.0 mg.
  • Subjects meet the following criteria for inclusion in this study. Subjects are males or females, 11-17 years of age inclusive. Subjects have histological evidence of NASH based on diagnostic liver biopsy, either during screening or from historical liver biopsy within the past six months, and elevated serum alanine aminotransferase (ALT) level of > 60 IU/L; have HbAlc of at least 9% or more, be otherwise stable health for the preceding 12 weeks; and have BMI of 40 kg/m or less. Subjects on insulin are on a single long acting insulin regime and the regime is stable for the past 4 weeks and remain stable throughout trial.
  • ALT serum alanine aminotransferase
  • subjects are not in the treatment or have been treated with NO donors, metformin, sildenafil, or thiazoladinediones within the preceding four weeks.
  • Female subjects are not lactating, pregnant or unwilling to practice adequate contraception for duration of the study.
  • Subjects do not have concomitant significant metabolic (other than diabetes), infectious, inflammatory, neoplastic or other non-liver disease. Excluded are also subjects who have currently or recent (within the previous four weeks) use of steroids or presence of concomitant disease requiring steroid use; or recent (within the previous twelve weeks) serious infection, such as pneumonia or urinary tract infection.
  • Subjects do not have weight loss of 5% or more in the last 3 months.
  • NAFLD non-alcoholic fatty liver disease
  • Subjects receiving the combination treatment are expected to have reduced serum alanine aminotransferase (ALT) activity during the 8-week treatment, and changes in other biomarker levels during 8-week treatment.
  • ALT serum alanine aminotransferase
  • subjects respond to the combination treatment are expected to have reduced AST level, reduced circulating K18 level, reduced blood lipids, reduced inflammation, and increased insulin sensitivity.
  • the placebo group is expected to maintain significant higher blood lipids, higher inflammation, higher ALT level, higher AST level and higher K18 level.
  • Example 5 Results of Interactive effect of Leucine and NO donors as a therapeutic for NAFLD/NASH [00288] We assessed the effects of leucine combined with L-Arginine or with
  • SNP Sodiumnitroprusside
  • L-Arginine The amino acid L-Arginine is a direct substrate of eNOS to produce NO. Concentrations between 0.5 mM to 28.7 mM are used in cell culture (primary cancer cells) to produce anti-pro liferative effects. Yao X, et al., Nitric oxide/cyclic guanosine monophosphate inducers sodium nitroprusside and L-arginine inhibit the proliferation of gastric cancer cells via the activation of type II cyclic guanosine monophosphate-dependent protein Kinase, Oncol Lett. (2015), 10(l):479-84. Lower concentrations (below 100 ⁇ ) are used for anti- inflammatory effects and NO production.
  • SNP Sodium nitroprusside
  • SNP is a direct NO donor clinically used to provide rapid lowering of blood pressure in hypertensive crises and it is used in clinical studies as the gold standard for endothelium-independent NO-mediated vasodilation.
  • Miller MR Recent developments in nitric oxide donor drugs, Br J Pharmacol. (2007), 151(3):305— 21.
  • High concentrations are (>800 ⁇ ) are neurotoxic in cell culture, while lower concentrations (0.1 ⁇ to 100 ⁇ ) are used for NO release.
  • hepatocytes exposed to high glucose media to stimulate lipid accumulation were unaffected by leucine or arginine individually. However, the combination of leucine and arginine resulted in about an 18-20% decrease in hepatocyte lipids.
  • hepatocytes exposed to high glucose media to stimulate lipid accumulation were unaffected by leucine or SNP individually.
  • the combination of leucine and SNP resulted in about an 18-20% decrease in hepatocyte lipids.

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Abstract

L'invention concerne des procédés utiles pour la réduction ou la prévention de la stéatose hépatique non alcoolique ou de la stéatose hépatique. De tels procédés peuvent consister à administrer, à un sujet en ayant besoin, un donneur d'oxyde nitrique (NO), un acide aminé ramifié sous forme d'acide aminé libre, ou un métabolite de celui-ci, un activateur du mécanisme d'action des sirtuines, un inhibiteur de la PDE5 seul ou toute combinaison de celui-ci. L'invention concerne également des compositions et des kits destinés à la mise en œuvre de l'un quelconque des procédés de l'invention.
PCT/US2017/049946 2016-09-02 2017-09-01 Donneurs d'oxyde d'azote et leurs utilisations WO2018045335A1 (fr)

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WO2021040813A1 (fr) * 2019-08-23 2021-03-04 NOTA Laboratories, LLC Systèmes de génération d'oxyde nitrique
CN113248530A (zh) * 2021-05-31 2021-08-13 浙江大学 一种活性氧响应的抗氧化一氧化氮供体、其制备和应用

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Publication number Priority date Publication date Assignee Title
WO2021040813A1 (fr) * 2019-08-23 2021-03-04 NOTA Laboratories, LLC Systèmes de génération d'oxyde nitrique
CN114667166A (zh) * 2019-08-23 2022-06-24 诺塔实验室有限责任公司 一氧化氮生成系统
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CN110836935A (zh) * 2019-11-25 2020-02-25 重庆柳江医药科技有限公司 测定甲磺司特原料药中3种基因毒性杂质的方法
CN110836935B (zh) * 2019-11-25 2022-01-28 重庆柳江医药科技有限公司 测定甲磺司特原料药中3种基因毒性杂质的方法
CN113248530A (zh) * 2021-05-31 2021-08-13 浙江大学 一种活性氧响应的抗氧化一氧化氮供体、其制备和应用
CN113248530B (zh) * 2021-05-31 2022-03-15 浙江大学 一种活性氧响应的抗氧化一氧化氮供体、其制备和应用

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