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  • 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
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Definitions

• the invention relates generally to the treatment of muscle loss in a mammal, and more particularly, to the administration of one or more branched chain amino acid(s) (BCAA), a BCAA precursor, a BCAA metabolite, a BCAA-rich protein, a protein manipulated to enrich the BCAA content or any combination thereof in the treatment of such muscle loss.
• BCAA branched chain amino acid
• the invention further relates to nutritional formulations suitable for such administration.
• Amino acids are the monomeric building blocks of proteins, which in turn comprise a wide range of biological compounds, including enzymes, antibodies, hormones, transport molecules for ions and small molecules, collagen, and muscle tissues.
• Amino acids are considered hydrophobic or hydrophilic, based upon their solubility in water, and, more particularly, on the polarities of their side chains.
• Amino acids having polar side chains are hydrophilic, while amino acids having non-polar side chains are hydrophobic.
• the solubilities of amino acids determines the structures of proteins. Hydrophilic amino acids tend to make up the surfaces of proteins while hydrophobic amino acids tend to make up the water-insoluble interior portions of proteins.
• a deficiency of one or more amino acids can cause a negative nitrogen balance.
• a negative nitrogen balance for example, is wherein more nitrogen is excreted than is administered. Such a condition can lead to disruption of enzymatic activity and the loss of muscle mass.
• cachexia is a severe body wasting condition characterized by marked weight loss, anorexia, asthenia, and anemia.
• Cachexia is a common feature of a number of illnesses, such as cancer, sepsis, chronic heart failure, rheumatoid arthritis, and acquired immune deficiency syndrome (AIDS).
• Other muscle wasting diseases and disorders are known, including, for example, sarcopenia, an age-related loss of muscle mass.
• PAF Proteolysis-lnducing Factor
• PIF proteolysis-inducing factor
• 15-HETE 15-hydroxyeicosatetraenoic acid
• 15-HETE has been shown to produce a significant increase in protein degradation and nuclear binding of the transcription factor NF- ⁇ B (a nuclear factor that binds the kappa immunoglobulin light chain gene enhancer in B cells).
• PIF RNA-dependent protein kinase
• met-tRNA binds to the 40s ribosomal subunit as a ternary complex with eukaryotic initiation factor 2 (elF2) and guanosine triphosphate (GTP).
• elF2 eukaryotic initiation factor 2
• GTP guanosine triphosphate
• the GTP bound to elF2 is hydrolyzed to guanosine diphosphate (GDP) and elF2 is released from the ribosomal subunit in a GDP-elF2 complex.
• GDP guanosine diphosphate
• the elF2 must then exchange the GDP for GTP to participate in another round of initiation. This occurs through the action of another eukaryotic initiation factor, elF2B, which mediates guanine nucleotide exchange on elF2.
• elF2B is regulated by the phosphorylation of elF2 on its alpha subunit, which converts it from
• elF4F a group of proteins collectively referred to as elF4F, a multisubunit complex consisting of elF4A (an RNA helicase), elF4B (which functions in conjunction with elF4A to unwind secondary structure in the 5' untranslated region of the mRNA), elF4E (which binds the m7GTP cap present at the 5' end of the mRNA), and elF4G (which functions as a scaffold for elF4E, elF4A, and the mRNA).
• elF4F a multisubunit complex consisting of elF4A (an RNA helicase), elF4B (which functions in conjunction with elF4A to unwind secondary structure in the 5' untranslated region of the mRNA), elF4E (which binds the m7GTP cap present at the 5' end of the mRNA), and elF4G
• the elF4F complex serves to recognize, unfold, and guide the mRNA to the 43s preinitiation complex.
• the availability of the elF4E for the elF4F complex formation appears to be regulated by the translational repressor elF4E-binding protein 1 (4E-BP1).
• 4E-BP1 competes with elF4G to bind elF4E and is able to sequester elF4E into an inactive complex.
• the binding of 4E-BP1 is regulated through phosphorylation by the kinase mammalian target of rapamycin (mTOR), where increased phosphorylation causes a decrease in the affinity of 4E-BP1 for elF4E.
• mTOR is activated by phosphorylation and inhibition of the tuberous sclerosis complex (TSC) 1-TSC2 complex via signaling through the phosphatidylinositol 3 kinase (PI3K) / serine/threonine kinase pathway (PI3K/AKT pathway).
• TSC tuberous sclerosis complex
• PI3K phosphatidylinositol 3 kinase
• PI3K/AKT pathway serine/threonine kinase pathway
• mTOR also phosphorylates p70S6 kinase, which phosphorylates ribosomal protein S6, which is believed to enhance the translation of mRNA with an uninterrupted string of pyrimidine residues adjacent to the 5' cap structure.
• Proteins encoded by such mRNA include ribosomal proteins, translation elongation factors, and poly-A binding proteins.
• anabolic factors such as insulin, insulin-like growth factors (IGFs), and amino acids increase protein synthesis and cause muscle hypertrophy.
• Branched chain amino acids BCAAs
• mitogenic stimuli such as insulin and BCAAs, signal via elF2.
• amino acid starvation results in an increased phosphorylation of elF2- ⁇ and a decrease in protein synthesis.
• PIF protein degradation via the NF- ⁇ B pathway. Therefore, it is plausible that inhibition of protein synthesis by PIF occurs via a common signaling initiation point, which then diverges into two separate pathways, one promoting protein degradation via NF- ⁇ B and the other inhibiting protein synthesis through mTOR and/or elF2.
• AKT is a serine/threonine kinase, also known as protein kinase B (PKB). Activation of AKT occurs through direct binding of the inositol lipid products of the PI3K to its pleckstrin homology domain. PI3K-dependent activation of AKT also occurs through phosphoinositide-dependent kinase (PDKI)-mediated phosphorylation of threonine 308, which leads to autophosphorylation of serine 473.
• PDKI phosphoinositide-dependent kinase
• NF- ⁇ B and AKT signaling pathways converge. Studies have shown that AKT signaling inhibits apoptosis in a variety of cell types in vitro, mediated by its ability to phosphorylate apoptosis-regulating components, including IKK, the
• AKT may be a downstream target of NF- ⁇ B. Overall, this suggests that AKT is involved in a catabolic pathway. Other data, however, suggest that AKT is also involved in anabolic processes through activation of mTOR and the consequent phosphorylation of p70S6 kinase and 4E-BP1, leading to an increase in protein synthesis.
• PKR is an interferon-induced, RNA-dependent serine/threonine protein kinase responsible for control of an antiviral defense pathway. PKR may be induced by forms of cellular stress other than interferon. Some evidence suggests that tumor necrosis factor (TNF)-alpha also acts through PKR. Interestingly, both interferon and TNF-alpha have been implicated as causative factors of cachectic states. Following interaction with activating stimuli (e.g., insulin, IGF, BCAAs), PKR has been reported to form ! homodimers and autophosphorylate.
• TNF tumor necrosis factor
• PKR is able to catalyze the phosphorylation of target substrates, the most well-characterised being the phosphorylation of Serine 51 on the elF2- ⁇ subunit.
• the elF2 then sequesters elF2B, a rate-limiting component of translation, resulting in the inhibition of protein synthesis.
• Recent studies suggest that PKR physically associates with the IKK complex and
• NF- ⁇ B-inducing kinase NIK
• NIK NF- ⁇ B-inducing kinase
• PKR-like ER-resident kinase (PERK) is another kinase that phosphorylates elF2- ⁇ and activates NF- ⁇ B. However, it is unlikely that PIF acts through this pathway, since
• PERK causes the release of IKK from NF- ⁇ B, but not its degradation.
• PIF PIF
• BCAAs Treatment of conditions such as cachexia often includes nutritional supplementation, and, in particular, amino acid supplementation, in an attempt to increase protein synthesis.
• the three BCAAs are valine, leucine, and isoleucine.
• leucine has been shown to function, not only as a protein building block, but also as an inducer of signal transduction pathways that modulate translation initiation.
• Our recent novel research suggests that all three of the BCAAs possess the ability to reduce protein degradation and enhance protein translation comparably.
• Cachexia is just one of the conditions, disorders, and diseases for which amino acid supplementation has proved beneficial.
• Amino acid supplementation has also been used to treat diabetes, hypertension, high levels of serum cholesterol and triglycerides, Parkinson's disease, insomnia, drug and alcohol addiction, pain, insomnia, and hypoglycemia.
• Supplementation with BCAAs in particular, has been used to treat liver disorders, including compromised liver function, including cirrhosis, gall bladder disorders, chorea and dyskinesia, and kidney disorders, including uremia.
• BCAA supplementation has also proved successful in the treatment of patients undergoing hemodialysis, resulting in improvements in overall health and mood.
• Leucine precursors such as pyruvate
• metabolites such as ⁇ -hydroxy- ⁇ -methylbutyrate and ⁇ -ketoisocaproate
• the amino acids that comprise skeletal muscle are in a constant state of flux where new amino acids, either coming from administration by enteral or parenteral routes or recirculated, are deposited as protein and current proteins are degraded. Loss of muscle mass can be the result of many factors including decreased rate of protein synthesis with normal degradation, increased degradation with normal synthesis or an exacerbation of both reduced synthesis and increased degradation. As a result, therapies aimed at increasing synthesis only address one-half of the problem in muscle wasting disease(s).
• the invention provides methods for treating muscle loss in an individual.
• the invention includes administering to an individual an effective amount of a branched chain amino acid (BCAA), a BCAA precursor, a BCAA metabolite, BCAA- rich protein, protein manipulated to enrich the BCAA content, or any combination thereof.
• BCAA branched chain amino acid
• the invention further provides nutritional products for such administration, including orally-administrable nutritional products.
• the invention provides a method of treating muscle loss in an individual, the method comprising: administering to the individual an effective amount of at least one of: a branched chain amino acid (BGAA); a BCAA precursor; and a BCAA metabolite, a BCAA-rich protein, a protein manipulated to enrich the BCAA content, wherein at least one of the BCAA, BCAA precursor, BCAA metabolite, BCAA-rich protein, and protein manipulated to enrich the BCAA content antagonizes protein catabolism.
• BGAA branched chain amino acid
• BCAA precursor a branched chain amino acid
• a BCAA metabolite a BCAA-rich protein
• a protein manipulated to enrich the BCAA content antagonizes protein catabolism.
• the invention provides an orally-administrable nutritional product comprising at least one of the following: a branched chain amino acid (BCAA); a BCAA precursor, a BCAA metabolite, a BCAA-rich protein, a protein manipulated to enrich the BCAA content, wherein at least one of the BCAA, BCAA precursor, BCAA metabolite, BCAA-rich protein, and protein manipulated to enrich the BCAA content antagonizes protein catabolism.
• a branched chain amino acid BCAA
• BCAA precursor branched chain amino acid
• BCAA metabolite a BCAA-rich protein
• a protein manipulated to enrich the BCAA content wherein at least one of the BCAA, BCAA precursor, BCAA metabolite, BCAA-rich protein, and protein manipulated to enrich the BCAA content antagonizes protein catabolism.
• FIG. 1 shows a graph of the depression of protein synthesis by proteolysis inducing factor (PIF) at various concentrations.
• FIG. 2 shows a graph of the effect of amino acids on the phosphorylation of elF2- ⁇ and PIF.
• FIG. 3 shows a graph of the effect of insulin and insulin-like growth factor 1 (IGF) on the phosphorylation of elF2- ⁇ of PIF.
• IGF insulin-like growth factor 1
• FIG. 4 shows the structure of an RNA-dependent protein kinase (PKR) inhibitor suitable for use in the present invention.
• PPK RNA-dependent protein kinase
• FIG. 5 shows a graph of the effect of the PKR inhibitor of FIG. 4 on the proteolytic activity of PIF.
• FIG. 6 shows a graph of the effect of the PKR inhibitor of FIG. 4 in reversing a PIF-mediated reduction in protein synthesis.
• FIG. 7 shows a graph of the effect of the PKR inhibitor of FIG. 4 on the proteolytic activity of Angiotensin II.
• FIG. 8 shows a graph of the effect of the PKR inhibitor of FIG. 4 in reversing an Angiotensin ll-mediated reduction in protein synthesis.
• FIG 9 shows an alternative mechanism of protein degradation caused by proteolysis inducing factor (PIF) and inhibited by branch chain amino acids, insulin and IGF-1.
• PAF proteolysis inducing factor
• FIG 10 shows a further alternative mechanism of protein degradation caused by proteolysis inducing factor (PIF) through activation of PKR and elF2 ⁇ that is inhibited by branch chain amino acids, insulin and IGF-1.
• PEF proteolysis inducing factor
• the invention provides methods and related products for the treatment of muscle loss in an individual. More specifically, the methods and products of the invention reduce muscle catabolism, particularly proteolysis-inducing factor (PIF) -mediated muscle catabolism.
• PEF proteolysis-inducing factor
• treatment refers to both prophylactic or preventive treatment and curative or disease-modifying treatment, including treatment of patients at risk of contracting a disease or suspected to have contracted a disease, as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition.
• treatment also refer to the maintenance and/or promotion of health in an individual not suffering from a disease but who may be susceptible to the development of an unhealthy condition, such as nitrogen imbalance or muscle loss. Consequently, an "effective amount” is an amount that treats a disease or medical condition in an individual or, more generally, provides a nutritional, physiological, or medical benefit to the individual.
• a treatment can be patient- or doctor- related.
• the terms “individual” and “patient” are often used herein to refer to a human, the invention is not so limited. Accordingly, the terms “individual” and “patient” refer to any mammal suffering from or at risk for a medical condition, such as muscle loss.
• branched chain amino acids BCAAs
• murine C 2 C 12 myotubes were exposed to PIF or Angiotensin Il in combination with amino acids (including BCAAs), insulin, insulin- like growth factor-1 (IGF-1), and a PKR inhibitor.
• PIF was extracted and purified from MAC16 tumors as described by Smith et aL, Effect of a Cancer Cachectic Factor on Protein Synthesis/Degradation in Murine C2C12 Myoblasts: Modulation by Eicosapentaenoic Acid, Cancer Research, 59:5507-13 (1999), which is hereby incorporated by reference.
• Protein degradation was determined using the method described by Whitehouse et al., Increased Expression of the Ubiquitin-Proteasome Pathway in Murine Myotubes by Proteolysis-lnducing Factor (PIF) is Associated with Activation of the Transcription Factor NF- ⁇ B, British Journal of Cancer, 89:1116-22
• FIG. 1 shows a graph of the depression of protein synthesis of PIF at increasing concentrations, measured in counts per minute (CPM) as a percentage of a control containing no PIF. A significant reduction in protein synthesis is noted, with a maximum depression of protein synthesis occurring at a PIF concentration of 4.2 nM.
• the measured proteolytic activity of PIF can be more specifically described as ubiquitin-like degradation activity.
• FIG. 2 shows a graph of the densitometry analysis of Western blots of phosphorylated elF2- ⁇ in C 2 Ci 2 myotubes incubated with PIF, leucine, isoleucine, valine, methionine, and arginine, both alone and in combination with PIF.
• the control sample was incubated only in phosphate buffered saline (PBS).
• PBS phosphate buffered saline
• BCAAs i.e., leucine, isoleucine, and valine
• methionine- and arginine-induced phosphorylation levels were greater than that of the control.
• these data show that all BCAAs are about equally effective in reducing PIF-induced phosphorylation of elF2- ⁇ .
• the phosphorylation levels resulting from isoleucine and valine incubation were not different from that observed with leucine incubation.
• FIG. 3 shows the results of similar experiments involving the incubation of insulin and IGF-1 , alone and in combination with PIF. Both insulin and IGF-1 significantly reduced elF2- ⁇ phosphorylation in the presence of PlF, compared to PIF alone. Thus, the ability of BCAAs to decrease PIF-mediated protein degradation may be supplemented or enhanced by the addition of insulin and/or IGF-1 or by treatments that increase the level of insulin and/or IGF-1.
• FIG. 4 shows the structure of a PKR inhibitor useful in both decreasing PIF- induced protein degradation and increasing protein synthesis which was used as a positive control of PKR inhibition.
• FIGS. 5-8 show the results of experiments involving the incubation of the PKR inhibitor in combination with either PIF or Angiotensin II.
• FIG. 5 it can be seen that while PIF increased protein degradation up to 87% when incubated alone, the addition of the PKR inhibitor reversed protein degradation levels back to about those of the control.
• FIG. 6 it can be seen that while PIF reduced protein synthesis up to about 25% when incubated alone, the addition of the PKR inhibitor reversed protein synthesis levels back to about those of the control.
• FIGS. 7 and 8 show similar results upon the incubation of the PKR inhibitor with Angiotensin II.
• Angiotensin increased protein degradation up to about 51%, compared to the control.
• the addition of the PKR inhibitor reversed this trend, maintaining protein degradation levels at about that of the control.
• Angiotensin Il reduced protein synthesis by about 40% compared to the control, while the addition of the PKR inhibitor maintained protein synthesis levels at about that of the control.
• the PKR inhibitor attenuated the actions of PIF and Angiotensin Il in both protein degradation and protein synthesis. This suggests that both PIF and Angiotensin Il mediate their effects through similar mechanisms and through a common mediator, likely involving PKR. More specifically, these results suggest that PIF activates PKR, which in turn causes phosphorylation of elF2- ⁇ , inhibiting the binding of initiator methionyl-tRNA (met-tRNA) to the 40s ribosomal subunit. BCAAs, insulin, and IGF-1 attenuated the phosphorylation of eIF2- ⁇ caused by PIF, further supporting the
• PKR can inhibit protein synthesis and activate NF- ⁇ B, which leads to protein degradation, PKR is likely an early component in the signaling pathway of PIF.
• PKR is involved in the regulation of 4E-BP1 phosphorylation.
• PIF does signal through PKR, it is likely that it can also reduce protein synthesis through PKR-mediated activation of the serine/threonine phosphatase PP2A, which can bring about the dephosphorylation of 4E-BP1 , which in turn sequesters elF4E into an inactive complex, preventing the formation of the 43s pre- initiation complex.
• FIG. 9 shows an alternative mechanism.
• PIF proteolysis inducing factor
• Ang II angiotensin Il
• PACT protein activator of interferon-induced protein kinase
• PKR cellular protein activator of PKR
• TNF- ⁇ tumor necrosis factor- ⁇
• FIG. 10 shows a further alternative mechanism.
• proteolysis inducing factor PIF
• angiotensin Il Ang II
• NF- ⁇ B may be activated by PIF or a
• NF- ⁇ B is not part of the same phosphorylation cascade despite having the same target to promote ubiquitin-taqging of proteins to be degraded.
• BCAAs may be employed to treat muscle loss in an individual by antagonizing protein catabolism mediated by PIF and/or Angiotensin Il through inhibiting the activation of PKR and/or elF2 ⁇ .
• each of the BCAAs is equally effective in such antagonization.
• the co-administration of insulin, IGF-1, and/or a PKR inhibitor, or the use of treatments to increase level of either or both of insulin and IGF-1 may increase the efficacy of BCAA treatments by further antagonizing protein catabolism, enhancing protein synthesis, or both.
• Nutritional products according to the invention may, therefore, include BCAAs, alone or in combination with insulin, IGF-1 , and/or a PKR inhibitor.
• BCAAs may be administered in their free forms, as dipeptides, as tripeptides, as polypeptides, as BCAA-rich protein, and/or as protein manipulated to enrich the BCAA content.
• Dipeptides, tripeptides and polypeptides may include two or more BCAAs. Where non- BCAAs are included in a dipeptide, tripeptide, or polypeptide preferred amino acids include alanine and glycine, but non-BCAAs may be any of the dispensable or indispensable (essential or non-essential) amino acids.
• preferred dipeptides include, but are not limited to, alanyl-leucine, alanyl-isoleucine, alanyl-valine, glycyl-leucine, glycyl-isoleucine, and glycyl-valine.
• Nutritional products according to the invention may similarly include precursors and/or metabolites of BCAAs, particularly precursors and/or metabolites of leucine, in addition to or in place of BCAAs.
• Such products may further include any number of additional ingredients, including, for example, a protein, a fiber, a fatty acid, a vitamin, a mineral, a sugar, a carbohydrate, a flavor agent, a medicament, and a therapeutic agent.
• the nutritional products of the present invention may be administered orally, via a feeding tube, or parenterally.
• Such products may be used in the treatment of an individual suffering from any number of muscle wasting diseases, disorders, or conditions, or any disease, disorder, or condition with which muscle loss is associated, including, for example, cachexia, cancer, tumor-induced weight loss, sepsis, chronic heart failure, rheumatoid arthritis, acquired immune deficiency syndrome (AIDS), sarcopenia, diabetes, hypertension, high levels of serum cholesterol, high levels of triglycerides, Parkinson's disease, insomnia, drug addiction, alcohol addiction, pain, insomnia, hypoglycemia, compromised liver function, including cirrhosis, gall bladder disorders, chorea, dyskinesia, and a kidney disorder, including uremia.
• cachexia cancer
• cancer tumor-induced weight loss
• sepsis chronic heart failure
• rheumatoid arthritis acquired immune deficiency syndrome
• AIDS acquired immune deficiency syndrome
• sarcopenia diabetes, hypertension, high levels of serum cholesterol, high levels of triglycerides

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• 2006
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