Compositions of selenium organic compounds and methods of use thereof

文档序号：293853 发布日期：2021-11-26 浏览：63次中文

阅读说明：本技术 硒有机化合物的组合物和其使用方法 (Compositions of selenium organic compounds and methods of use thereof ) 是由 R·鲍威尔兰子鉴 A·伊安尼库里斯于 2014-03-14 设计创作，主要内容包括：本申请涉及硒有机化合物的组合物和其使用方法。具体而言,本申请涉及包含硒化合物的组合物和使用所述组合物增强线粒体功能或治疗线粒体功能障碍的方法,所述硒化合物例如5’-甲基硒代腺苷、Se-腺苷-L-高半胱氨酸、γ-谷氨酰基-甲基硒代半胱氨酸、式(I)的化合物、式(II)的化合物、式(III)的化合物和其组合。(The present application relates to compositions of selenium organic compounds and methods of use thereof. In particular, the present application relates to compositions comprising selenium compounds, such as 5' -methylselenoadenosine, Se-adenosyl-L-homocysteine, gamma-glutamyl-methylselenocysteine, and methods of using the compositions to enhance mitochondrial function or treat mitochondrial dysfunctionA compound of formula (I), a compound of formula (II), a compound of formula (III), and combinations thereof.)

1. A method for treating alzheimer's disease comprising:

administering to the subject an effective amount of a composition comprising a compound selected from the group consisting of 5' -methylselenoadenosine, a compound of formula (I), and mixtures thereof.

2. A method for inhibiting B amyloid accumulation in one or more neuronal cells, comprising:

administering to the one or more neuronal cells an effective amount of a composition comprising a compound selected from the group consisting of 5' -methylselenoadenosine, a compound of formula (I), and mixtures thereof, wherein the effective amount inhibits B amyloid accumulation in neuronal cells compared to neuronal cells not treated with the composition.

3. A method for inhibiting tau phosphorylation in one or more neuronal cells, comprising:

administering to the one or more neuronal cells an effective amount of a composition comprising a compound selected from the group consisting of 5' -methylselenoadenosine, a compound of formula (I), and mixtures thereof, wherein the effective amount inhibits tau phosphorylation in neuronal cells compared to neuronal cells not treated with the composition.

4. The method of any one of claims 1-3, wherein the compound is isolated.

5. The method of claim 4, wherein the compound is purified.

6. The method of any one of claims 1-5, wherein the composition comprises 5' -methylselenoadenosine.

7. The method of any one of claims 1-6, wherein the composition excludes one or more of glutamylselenocysteine, methionine, or selenomethionine.

8. The method of any one of claims 1-7, wherein the effective amount is 200 micrograms per day or less.

9. The method of any one of claims 1-8, wherein the composition is administered once daily.

10. The method of any one of claims 1-9, wherein the 5' -methylselenoadenosine or the compound of formula (I) is a selenoglycoside.

11. A method for enhancing mitochondrial function in one or more cells selected from the group consisting of skeletal muscle cells, neuronal cells, and combinations thereof, the method comprising:

administering to the one or more cells an effective amount of a composition comprising a compound selected from the group consisting of 5' -methylselenoadenosine, Se-adenosyl-L-homocysteine, gamma-glutamyl-methylseleno-cysteine, a compound of formula (I), a compound of formula (III), and combinations thereof, wherein the effective amount enhances mitochondrial function as compared to cells not treated with the composition.

12. A method for enhancing mitochondrial function in one or more hepatocytes, comprising:

administering to the one or more hepatocytes an effective amount of a composition comprising at least three different compounds selected from the group consisting of 5' -methylselenoadenosine, Se-adenosyl-L-homocysteine, gamma-glutamyl-methylseleno-cysteine, a compound of formula (I), and a compound of formula (III), wherein the effective amount enhances mitochondrial function as compared to cells not treated with the composition.

13. A method of modulating glucose metabolism in one or more cells selected from the group consisting of hepatocytes, skeletal muscle cells, and a combination thereof, the method comprising:

administering to the one or more cells an effective amount of a composition comprising at least three different compounds selected from the group consisting of 5' -methylselenoadenosine, Se-adenosyl-L-homocysteine, gamma-glutamyl-methylseleno-cysteine, a compound of formula (I), and a compound of formula (III), wherein the effective amount modulates glucose metabolism in the one or more cells compared to cells not treated with the composition.

14. A method of reducing expression of a glucose 6 phosphatase complex in one or more hepatocytes, the method comprising:

administering to the one or more cells an effective amount of a composition comprising at least three different compounds selected from the group consisting of 5' -methylselenoadenosine, Se-adenosyl-L-homocysteine, gamma-glutamyl-methylseleno-cysteine, a compound of formula (I), and a compound of formula (III), wherein the effective amount inhibits expression of glucose 6 phosphatase in one or more hepatocytes compared to cells not treated with the composition.

15. A composition comprising a compound of formula (I):

Or a pharmaceutically acceptable salt, hydrate or prodrug thereof, wherein

R₁Is H, acyl, alkyl, alkenyl, alkynyl, aralkyl, carboxyl, cycloalkyl, C (O) R ', C (O) OR ', wherein R ' is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, OR heterocyclyl; or R₁And R₂Together form a heterocyclic ring having 4-8 ring members and at least one heteroatom selected from oxygen or nitrogen;

R₂is H, acyl, alkyl, alkenyl, alkynyl, aralkyl, carboxy, cycloalkyl, C (O) R ', C (O) OR ', wherein R ' is selected from alkyl, cycloalkyl, aryl, aralkyl, OR heterocyclyl; or R₁And R₂Together form a heterocyclic ring having 4-8 ring members and at least one heteroatom selected from oxygen or nitrogen;

R₃is H, acyl, alkyl, alkenyl, alkynyl, aralkyl, cycloalkyl, carboxyl or C-amido; or R₃And R₄Together with the atoms to which they are attached form a heterocyclic ring having 4-8 ring members and at least one heteroatom selected from oxygen or nitrogen;

R₄is H, acyl, alkyl, alkenyl, alkynyl, aralkyl, cycloalkyl, carboxyl or C-amido; or R₃And R₄Together with the atoms to which they are attached form a ring having 4-8 ring members and at least one heteroatom selected from oxygen or nitrogen The heterocyclic ring of (1);

R₅is oxo, hydroxy, alkyl, alkenyl, alkynyl, OR' OR absent; wherein R' is selected from alkyl, alkenyl, alkynyl, cycloalkyl, aryl or aralkyl;

R₆is oxo, hydroxy, alkyl, alkenyl, alkynyl, OR' OR absent; wherein R' is selected from alkyl, alkenyl, alkynyl, cycloalkyl, aryl or aralkyl;

R₇is H, alkyl, alkenyl, alkynyl, ketone, amino alcohol, amino acid, OR ', Se-R', S-R ', wherein R' is selected from H, alkyl, cycloalkyl, aryl, aralkyl, OR heterocyclyl; and

R₈is hydrogen, azido, alkyl, alkenyl, alkynyl.

16. A composition comprising a compound of formula (II):

or a pharmaceutically acceptable salt, hydrate or prodrug thereof, wherein

R₂is H, acyl, alkyl, alkenyl, alkynyl, aralkyl, carboxy, cycloalkyl, C (O) R ', C (O) OR ', wherein R ' is selected from alkyl, cycloalkyl, aryl, aralkyl, OR heterocyclyl; or R ₁And R₂Together form a heterocyclic ring having 4-8 ring members and at least one heteroatom selected from oxygen or nitrogen;

R₃is H, acyl, alkyl, alkenyl, alkynyl, aralkyl, cycloalkyl, carboxyl or C-amido; or R₃And R₄Together with the atoms to which they are attached form a ring having 4-8 atomsA heterocyclic ring of ring members and at least one heteroatom selected from oxygen or nitrogen;

R₄is H, acyl, alkyl, alkenyl, alkynyl, aralkyl, cycloalkyl, carboxyl or C-amido; or R₃And R₄Together with the atoms to which they are attached form a heterocyclic ring having 4-8 ring members and at least one heteroatom selected from oxygen or nitrogen;

R₅is oxo, hydroxy, alkyl, alkenyl, alkynyl, OR' OR absent; wherein R' is selected from alkyl, alkenyl, alkynyl, cycloalkyl, aryl or aralkyl;

R₆is oxo, hydroxy, alkyl, alkenyl, alkynyl, OR' OR absent; wherein R' is selected from alkyl, alkenyl, alkynyl, cycloalkyl, aryl or aralkyl;

R₈is hydrogen, azido, alkyl, alkenyl, alkynyl;

R₉is H, acyl, alkyl, alkenyl, alkynyl, aralkyl, carboxy, cycloalkyl, C (O) R ', C (O) OR ', wherein R ' is alkyl, cycloalkyl, aryl, aralkyl, OR heterocyclyl; or R ₉And R₁₀Together form a heterocyclic ring having 4-8 ring members and at least one heteroatom selected from oxygen or nitrogen;

R₁₀is H, acyl, alkyl, alkenyl, alkynyl, aralkyl, carboxy, cycloalkyl, C (O) R ', C (O) OR ', wherein R ' is selected from alkyl, cycloalkyl, aryl, aralkyl, OR heterocyclyl; or R₉And R₁₀Together form a heterocyclic ring having 4-8 ring members and at least one heteroatom selected from oxygen or nitrogen; and

R₁₁is OH, OR, alkoxy, aralkoxy, OR amino, wherein R is selected from alkyl, cycloalkyl, aryl, aralkyl, heterocyclyl, OR a pharmaceutically acceptable salt OR inner salt.

17. A composition comprising a compound of formula (III):

or a pharmaceutically acceptable salt, hydrate or prodrug thereof, wherein

R₂is H, acyl, alkyl, alkenyl, alkynyl, aralkyl, carboxy, cycloalkyl, C (O) R ', C (O) OR ', wherein R ' is selected from alkyl, cycloalkyl, aryl, aralkyl, OR heterocyclyl; or R ₁And R₂Together form a heterocyclic ring having 4-8 ring members and at least one heteroatom selected from oxygen or nitrogen;

R₃is OH, OR, alkoxy, aralkoxy, OR amino, wherein R is selected from alkyl, cycloalkyl, aryl, aralkyl, heterocyclyl, OR a pharmaceutically acceptable salt OR inner salt;

R₄is H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocyclyl, or a pharmaceutically acceptable salt or inner salt;

R₅is oxo, hydroxy, alkyl, alkenyl, alkynyl, OR' OR absent; wherein R' is selected from alkyl, alkenyl, alkynyl, cycloalkyl, aryl or aralkyl;

R₆is oxo, hydroxy, alkyl, alkenyl, alkynyl, OR' OR absent; wherein R' is selected from alkyl, alkenyl, alkynyl, cycloalkyl, aryl or aralkyl; and

R₇is H, alkyl, alkenyl, alkynyl, ketone, OR ', Se-R', S-R ', wherein R' is selected from H, alkyl, cycloalkyl, aryl, aralkyl OR heterocyclyl.

Technical Field

The present application relates to selenium organic compositions and compositions of compounds, and methods of their use in various biological pathways to enhance mitochondrial function, treat diseases, and inhibit or enhance specific processes in specific types of animal and human cells.

Background

Selenium (Se) is an essential trace element that plays a key role in many biological processes, such as reproduction, thyroid hormone metabolism, DNA synthesis, and protection from oxidative damage and infection. Selenium is incorporated at the catalytic site of various selenium-dependent enzymes, such as glutathione peroxidase (GPx), thioredoxin reductase and a sulfoxy methionine reductase. These selenolases promote regulation of metabolic activity, immune function, antioxidant defense, intracellular redox regulation and mitochondrial function.

The organelles known as the mitochondria ("MT") are the major source of energy in cells of higher organisms. Mitochondria provide direct and indirect biochemical regulation of a wide range of cellular processes of respiration, oxidation, and metabolism. These processes include Electron Transport Chain (ETC) activity, which drives oxidative phosphorylation to produce metabolic energy in the form of Adenosine Triphosphate (ATP), and which is also the basis of important mitochondrial function in intracellular calcium homeostasis. Mitochondrial respiration occurs on the inner mitochondrial membrane by electron flow through an electron transport system comprising four complexes (complexes I, II, III and IV), and another complex (complex V) which serves as the site of ATP synthesis (ATP synthase). Impairment or reduction of the activity of any complex disrupts electron flow and may lead to mitochondrial respiratory dysfunction (see, e.g., Schildgen et al, Exp Hematol 2011;39: 666-.

Mitochondrial dysfunction leading to cell death, reactive oxygen species production, increased oxidative DNA damage, increased autophagy, and loss of mitochondrial membrane potential is associated with conditions such as diabetes, obesity, age-related neurodegeneration (including alzheimer's disease), stroke, insulin resistance, and atherosclerosis. Inorganic forms of selenium, sodium selenite, have been shown to affect mitochondrial function in some cases. Mehta et al show that the marker for mitochondrial biogenesis, PGC1a, is increased in ischemic brain tissue and sodium selenite further increases PGC1a after ischemia and recirculation. (Mehta et al, BMC Neuroscience 201213: 79). Tirosh et al show that high, but not moderate, doses of sodium selenite prevent hapten-induced mitochondrial function damage due to hapten-induced inflammation in colon tissue. (Tirosh et al Nutrition 200723: 878). These results indicate that inorganic selenium may affect mitochondrial function in cells suffering from damage. However, some studies have found that sodium selenite is less bioavailable than other forms of selenium, questioning its efficacy. (Rider et al, J Anim Physiol Anim Nutr (Berl) 201094 (1): 99-110).

Furthermore, the results in the literature indicate that different chemical forms of selenium have different biological activities. For example, selenozolidine is more effective than selenomethionine in reducing the number of lung tumors. (Poerschke et al, J Biochem Molecular biology 201226: 344). Barger et al show that mice fed different sources of selenium, such as selenomethionine, sodium selenite, and selenized yeast, have different effects on gene expression and specific functional pathways on mitochondrial structure and function. (Barger et al, Genes and Nutrition 20127: 155).

Because the biological activity and availability of different chemical forms of selenium vary significantly, there is a need to identify chemical forms of selenium and characterize their effect on biological processes. Characterization of these effects on biological processes can lead to medical modulation of important biological processes to prevent or combat diseases, such as those associated with mitochondrial dysfunction. Diseases associated with mitochondrial dysfunction can be prevented or treated by administering a specific chemical form of selenium that reduces mitochondrial dysfunction or upregulates mitochondrial function in one or more types of animal or human cells. One explanation for the variation in biological activity may be that different forms of selenium have different effects on biological pathways at the molecular level.

Disclosure of Invention

The present application relates to selenium-organic compounds, compositions, and methods of using the compounds and compositions. The compounds include 5' -methylselenoadenosine ("compound C"), Se-adenosyl-L-homocysteine ("compound D"), gamma-glutamyl-methylselenocysteine ("compound E"), compounds of formula I, compounds of formula II, compounds of formula III, and combinations thereof. As described herein, the compositions and combinations thereof are useful for enhancing mitochondrial function, treating mitochondrial dysfunction, treating alzheimer's disease, and regulating glucose metabolism in a tissue-specific and tissue-appropriate manner.

In one aspect of the application, different chemical forms of organic selenium are identified as being biologically active. The selenium-containing compounds as described herein may be obtained from selenized yeast or may be chemically synthesized as described herein. Selenized yeast contains many selenium and sulfur compounds, but not all selenium compounds in selenized yeast affect biological processes. Furthermore, mixtures of selenium and sulfur compounds in selenized yeast have been shown to inhibit each other or negatively affect biological processes.

Another aspect of the present application provides an analog or derivative of the bioactive selenium compound described herein. Analogs and/or derivatives of selenium-containing compounds can be synthetically prepared. In some embodiments, the analogs have increased stability, reduced oxidation, increased half-life, and/or target the compound to a particular tissue.

In another aspect of the present application, different chemical forms of selenium are shown to have different tissue specificities (e.g., some compounds are active on neuronal cells, but not on hepatocytes). In addition, the selenium-containing composition can activate a transcriptional activator in one cell type, while it inactivates the transcriptional activator in a different cell type. These different activities and tissue specificities of different selenium containing compounds and combinations thereof are surprising and unexpected.

One aspect of the present application provides a composition comprising a compound selected from the group consisting of 5' -methylselenoadenosine, Se-adenosyl-L-homocysteine, gamma-glutamyl-methylselenocysteine, a compound of formula (I), a compound of formula II, a compound of formula (III), and combinations thereof. In further embodiments, one or more of these compounds may be isolated and/or purified.

In other embodiments, the composition may exclude one or more of 5' -methylthioadenosine, S-adenosyl-L-homocysteine, gamma-glutamyl-methyl-cysteine, or glutamyl selenocysteine, as one or more of these compounds may not be necessary for the composition, or inhibit other compounds in the composition.

Another aspect of the present application provides methods of enhancing mitochondrial function, treating mitochondrial dysfunction, treating alzheimer's disease, and/or modulating glucose metabolism using the compositions described herein.

In embodiments, the method for treating alzheimer's disease comprises: administering to the subject an effective amount of a composition comprising 5' -methylselenoadenosine, a compound of formula (I), or a mixture thereof. In other embodiments, one or more of these compounds may be isolated and/or purified.

In still other embodiments, the method for treating alzheimer's disease comprises: administering to the subject an effective amount of a composition comprising at least three different compounds selected from the group consisting of 5' -methylselenoadenosine, Se-adenosyl-L-homocysteine, gamma-glutamyl-methylselenocysteine, a compound of formula (I), and a compound of formula (III). In embodiments of the invention, one or more of these compounds may be isolated and/or purified.

In other embodiments, a method for inhibiting B amyloid accumulation in one or more neuronal cells comprises: administering to the one or more neuronal cells an effective amount of a composition comprising a compound selected from the group consisting of 5' -methylselenoadenosine, a compound of formula (I), and mixtures thereof, wherein the effective amount inhibits B amyloid accumulation in the one or more neuronal cells compared to neuronal cells not treated with the composition. In further embodiments, one or more of these compounds may be isolated and/or purified.

In another embodiment, a method for inhibiting B amyloid accumulation in one or more neuronal cells comprises: administering to one or more neuronal cells an effective amount of a composition comprising at least three different compounds selected from the group consisting of 5' -methylselenoadenosine, Se-adenosyl-L-homocysteine, gamma-glutamyl-methylseleno-cysteine, a compound of formula (I), and a compound of formula (III), wherein the effective amount inhibits B amyloid accumulation in neuronal cells compared to neuronal cells not treated with the composition. In further embodiments, one or more of these compounds may be isolated and/or purified.

In embodiments, a method for inhibiting tau phosphorylation in one or more neuronal cells comprises: administering to the one or more neuronal cells an effective amount of a composition comprising a compound selected from the group consisting of 5' -methylselenoadenosine, a compound of formula (I), and mixtures thereof, wherein the effective amount inhibits tau phosphorylation in the one or more neuronal cells as compared to neuronal cells not treated with the composition. In further embodiments, one or more of these compounds may be isolated and/or purified.

In other embodiments, a method for inhibiting tau phosphorylation in one or more neuronal cells comprises: administering to the one or more neuronal cells an effective amount of a composition comprising at least three different compounds selected from the group consisting of 5' -methylselenoadenosine, Se-adenosyl-L-homocysteine, gamma-glutamyl-methylselenocysteine, a compound of formula (I), and a compound of formula (III), wherein the effective amount inhibits tau phosphorylation in neuronal cells compared to neuronal cells not treated with the composition. In further embodiments, one or more compounds may be isolated and/or purified.

In embodiments, the composition comprises 5' -methylselenoadenosine or a compound of formula (I) or a selenoglycoside thereof. In still other embodiments, the composition may exclude glutamylselenocysteine, methionine or selenomethionine because of their possible inhibitory effect in the composition on the active compounds in the composition.

In some embodiments, the effective amount administered to the subject is 200 micrograms per day or less. In embodiments, the compositions of the present invention are administered to a subject once a day.

In another aspect, a method for enhancing mitochondrial function in one or more cells selected from the group consisting of skeletal muscle cells, neuronal cells, and combinations thereof comprises: administering to the one or more cells an effective amount of a composition comprising a compound selected from the group consisting of 5' -methylselenoadenosine, Se-adenosyl-L-homocysteine, gamma-glutamyl-methylseleno-cysteine, a compound of formula (I), a compound of formula (III), and combinations thereof, wherein the effective amount enhances mitochondrial function as compared to cells not treated with the composition. In further embodiments, one or more compounds may be isolated and/or purified.

In other embodiments, a method for enhancing mitochondrial function in one or more hepatocytes comprises: administering to the one or more hepatocytes an effective amount of a composition comprising at least three different compounds selected from the group consisting of 5' -methylselenoadenosine, Se-adenosyl-L-homocysteine, gamma-glutamyl-methylseleno-cysteine, a compound of formula (I), and a compound of formula (III), wherein the effective amount enhances mitochondrial function as compared to cells not treated with the composition. In further embodiments, one or more compounds may be isolated and/or purified.

In another aspect, a method of modulating glucose metabolism in one or more cells selected from the group consisting of hepatocytes, skeletal muscle cells, and mixtures thereof, the method comprising: administering to the one or more cells an effective amount of a composition comprising at least three different compounds selected from the group consisting of 5' -methylselenoadenosine, Se-adenosyl-L-homocysteine, gamma-glutamyl-methylseleno-cysteine, a compound of formula (I), and a compound of formula (III), wherein the effective amount modulates glucose metabolism as compared to cells not treated with the composition. In further embodiments, one or more compounds may be isolated and/or purified.

In embodiments, a method of reducing the expression of a glucose 6 phosphatase complex in one or more hepatocytes, comprising: administering to the one or more cells an effective amount of a composition comprising at least three different compounds selected from the group consisting of 5' -methylselenoadenosine, Se-adenosyl-L-homocysteine, gamma-glutamyl-methylseleno-cysteine, a compound of formula (I), and a compound of formula (III), wherein the effective amount inhibits the expression of glucose 6 phosphatase compared to cells not treated with the composition. In further embodiments, one or more compounds may be isolated and/or purified.

Drawings

The accompanying drawings incorporated in and forming a part of the specification illustrate several aspects of the present invention and, together with the description, serve to explain the principles of the invention. The brief description of the drawings is as follows:

figure 1 shows an increase in mitochondrial ("MT") potential (fluorescence) when HEK293T kidney cells were treated with 5' -methylselenoadenosine ("compound C") instead of Se-adenosyl-L-homocysteine ("compound D"). Images were captured under a fluorescence microscope at the same exposure time and magnification.

Figure 2 shows an increase in mitochondrial ("MT") potential in skeletal muscle C2C12 cells upon treatment with compounds C (5' -methylselenoadenosine) and D (Se-adenosyl-L-homocysteine). The sulfur analogs H (5' -methylthioadenosine) and I (S-adenosyl-L-homocysteine) reduced mitochondrial potential at all concentrations.

Figure 3 shows transient increases in mitochondrial ("MT") potential at 6 and 24 hours in IMR-32 human neuronal cells treated with compounds C (5' -methylselenoadenosine), D (Se-adenosyl-L-homocysteine), and E (γ -glutamyl-methylseleno-cysteine), or a combination thereof. Their sulfur analogs H (5' -methylthioadenosine), I (S-adenosyl-L-homocysteine), J (γ -glutamyl-methyl-cysteine), and combinations thereof also show a transient increase in mitochondrial function. Data were normalized by fluorescence intensity of stained nuclei. Denotes with control PThe value is obtained.

Figure 4 shows that repeated treatment of C (5 '-methylselenoadenosine), D (Se-adenosyl-L-homocysteine) and E (γ -glutamyl-methylseleno-cysteine) or their sulfur analogs H (5' -methylthioadenosine), I (S-adenosyl-L-homocysteine), J (γ -glutamyl-methyl-cysteine) increased Mitochondrial (MT) potential in IMR-32 neuronal cells. Cells were treated once with compound for 48 hours (hr.) (upper panel) or twice with compound for 48 hours (hr.) (lower panel). Data were normalized by fluorescence intensity of stained nuclei.

Figure 5 shows that compounds C (5' -methylselenoadenosine), D (Se-adenosyl-L-homocysteine) and E (γ -glutamyl-methylselenocysteine), or a combination thereof, lack toxic effects on the viability of human IMR-32 neuronal cells (expressed by OD490 nm) over a 72 hour period. In contrast, the sulfur analogs H (5' -methylthioadenosine), I (S-adenosyl-L-homocysteine), J (γ -glutamyl-methyl-cysteine), and combinations thereof showed a slight decrease in viability over the 72 hour period. The results are shown as mean + sem, n = 8.

Figure 6 shows the restoration of mitochondrial function by compounds D and E in rat cortical cells stressed by 10 micromolar calcium. The graph shows the respiration map of normal mitochondria (highest mitochondria), with final OCR being the measured distance between the end of the map line and the X-axis. The lowest mitochondria indicated inhibition of mitochondrial respiration by 10 micromolar calcium. The middle two lines represent mitochondrial respiration in the presence of compound C or D and 10 micromolar calcium.

Figure 7 shows a significant increase in Mitochondrial (MT) potential in hepatocytes of AML-12 mice treated with a combination of selenium compounds C (5 '-methylselenoadenosine), D (Se-adenosyl-L-homocysteine), and E (γ -glutamyl-methylseleno-cysteine) (150 ppb each) at 6 and 24 hours compared to the combination of H (5' -methylthioadenosine), I (S-adenosyl-L-homocysteine), and J (γ -glutamyl-methyl-cysteine). Data were normalized by fluorescence intensity of stained nuclei. Shown in bar chartPValues were determined by comparing the CDE group with the control or HIJ group.

Figure 8 shows significant expression of mitochondrial uncoupling protein 2 (Ucp2) in AML-12 mouse hepatocytes and downregulation of its expression by the combination of compounds C (5' -methylselenoadenosine), D (Se-adenosyl-L-homocysteine) and E (γ -glutamyl-methylselenocysteine) (150 ppb each). (A) Relative expression of mitochondrial uncoupling proteins 1 (Ucp1) and Ucp2 in normal AML-12 cells (after 6 hours of treatment with aqueous vehicle). n =4 as shown in the column. (B) The combination of C (5' -methylselenoadenosine), D (Se-adenosyl-L-homocysteine) and E (γ -glutamyl-methylselenocysteine) had no effect on Ucp1 expression. (C) The combination of C (5' -methylselenoadenosine), D (Se-adenosyl-L-homocysteine) and E (γ -glutamyl-methylselenocysteine) down-regulated Ucp2 expression. Data are expressed as the average of the number of samples indicated in each column + sem. The different letters (a and b) in the histogram mean the significant difference in these groups: ( P < 0.05)。

Figure 9 shows that compounds C (5 '-methylselenoadenosine), D (Se-adenosyl-L-homocysteine), E (γ -glutamyl-methylselenocysteine), and combinations thereof or their sulfur analogs H (5' -methylthioadenosine), I (S-adenosyl-L-homocysteine), J (γ -glutamyl-methyl-cysteine), and combinations thereof, have no toxic effect on the viability of AML-12 mouse hepatocytes (indicated by OD490 nm).

FIG. 10 shows the down-regulation of Ucp2, Uc in human IMR-32 neuronal cells following treatment with a combination of C (5' -methylselenoadenosine), D (Se-adenosyl-L-homocysteine) and E (gamma-glutamyl-methylseleno-cysteine) (150 ppb each) compoundsp3 and Presenilin (PSEN). (A) Ucp2 mRNA expression at 6 and 24 hours. (B) Ucp3 mRNA expression at 6 and 24 hours. (C) Relative PSEN1 and PSEN2 mRNA levels at 6 and 24 hours in human IMR-32 neuronal cells treated with vehicle (water). (D) PSEN mRNA expression at 6 and 24 hours after normalization to the amount of actin β (ACTB) mRNA levels in human IMR-32 neuronal cells treated with a combination of C (5 '-methylselenoadenosine), D (Se-adenosyl-L-homocysteine), E (γ -glutamyl-methylseleno-cysteine) (150 ppb each) or a combination of H (5' -methylthioadenosine), I (S-adenosyl-L-homocysteine) and J (γ -glutamyl-methyl-cysteine) (150 ppb each). (E) PSEN2 expression at 6 and 24 hours after normalization to (ACTB) mRNA levels in human IMR-32 neuronal cells treated with a combination of C (5 '-methylselenoadenosine), D (Se-adenosyl-L-homocysteine), and E (γ -glutamyl-methylseleno-cysteine) or a combination of H (5' -methylthioadenosine), I (S-adenosyl-L-homocysteine), and J (γ -glutamyl-methyl-cysteine). Data are expressed as mean + sem of 3-4 samples/group. Different letters (a and b) and different numbers (1 and 2) in the histogram mean significant differences in these groups: ( P < 0.05)。

Figure 11 shows that gamma secretase complex PSEN and Nicastrin are targets for compound C (5' -methylselenoadenosine), as determined by western blot and real-time polymerase chain reaction ("RT-PCR") analysis. (A) Western blot analysis of various proteins in human IMR-32 neuronal cells critical for plaque formation in Alzheimer's Disease (AD) treated with 150ppb of the compounds C (5' -methylselenoadenosine), D (Se-adenosyl-L-homocysteine) or E (γ -glutamyl-methylseleno-cysteine) for 6 and 24 hours. (B-C) quantitative analysis of the levels of (B) PSEN and (C) Nicastrin proteins in the above Western blot (24 hours after treatment with the compounds listed, right panel). Data are expressed as the mean of 3 samples + sem. (D-G) quantitative RT-PCR analysis of (D-E) PSEN and (F-G) Nicastrin expression in human IMR-32 neuronal cells treated (D, F) 6 and (E, G) 24 hr with aqueous vehicle (control) and the compounds listed. Data are expressed as the mean + sem of 4 samples. Differences in histogramsThe letters (a and b, a and c or b and c) mean the significant difference between the two groups: (P <0.05). The letters "a, b" indicate no significant difference from a or b.

FIG. 12 shows that compound C (5' -methylselenoadenosine) is a novel inhibitor of Tau phosphorylation, and a glycogen synthase kinase 3 β ("GSK 3 b") down-regulator, as determined by Western blot and RT-PCR analysis. (A) Western blot analysis of various proteins (critical for tangle formation in AD) in human IMR-32 neuronal cells treated with 150ppb of the compounds C (5' -methylselenoadenosine), D (Se-adenosyl-L-homocysteine) or E (γ -glutamyl-methylseleno-cysteine) for 6 and 24 hours. (B-E) quantitative analysis of the levels of phosphorylated Tau at the serine residue at position 396 (B) ("pTau S396") and serine residue at position 400, (C) threonine residue at position 403 and phosphorylated Tau at the serine residue at position 404 ("pTau S400/T403/S404"), (D) total Tau and (E) combined pTau S396 and pTau S400/T403/S404/total Tau in the above Western blot (after 24 hours of treatment, right panel). Data are expressed as the mean of 3 samples + sem. (F) Quantitative analysis of GSK3b protein levels in the above Western blots of IMR-32 cells treated with either aqueous vehicle (control) or compound for 24 hours. Data are expressed as the mean of 3 samples + sem. (G-H) quantitative RT-PCR analysis of GSK3b mRNA expression in human IMR-32 neuronal cells treated with aqueous vehicle (control) and the compounds listed (G) 6 and (H) for 24 hours. Data are expressed as the mean + sem of 4 samples. Different letters in the histogram (a and b, a and c, or b and c) mean significant differences between the two groups: ( P <0.05). The letters "a, b" or "b, c" mean no significant difference from a or b or c.

Figure 13 shows a decrease in FOXO phosphorylation and an increase in PGC1a protein expression in human IMR-32 neuronal cells caused by the combination of compounds C (5' -methylselenoadenosine), D (Se-adenosyl-L-homocysteine) and E (γ -glutamyl-methylseleno-cysteine), each compound 150 ppb.

FIG. 14 shows Western blot analysis of various other signal transduction molecules, including the compound C (5' -methylselenium)Phophorylated forkhead box protein O4 phosphorylated on threonine 28 ("pFOXO 4T 28"), forkhead box protein O4 ("FOXO 4"), phosphorylated murine thymoma virus oncogene homolog 1 on threonine 308 ("pakt 308"), phosphorylated murine thymoma virus oncogene homolog 1 on serine 471 ("pAktS 471"), Akt, and peroxisome proliferator-activated receptor gamma co-activator 1 α ("PGC 1 a"), 150ppb of each compound in adenosine), D (Se-adenosyl-L-homocysteine), or E (γ -glutamyl-methylselenocysteine) -treated IMR-32 neuronal cells. (A) Western blot photograph. (B-C) quantitative analysis of phosphorylated forkhead box protein O4 (pFOXO 4T 28) on threonine at position 28 in the above-described Western blot of AML-12 cells treated for 24 hours (B) 6 and (C) with the listed compounds. Data are expressed as the mean of 3 samples + sem. The different letters (a and b) in the histogram mean that there are significant differences between the two groups: ( P <0.05). The letters "a, b" indicate no significant difference from a or b.

Figure 15 shows western blot analysis of various other signaling molecules including FOXOs, PDK1, AKT, Gsk3a/b, 4EBP1, Elf2be, and PGC1a in mouse hepatic AML-12 cells treated with a combination of C (5' -methylselenoadenosine), D (Se-adenosyl-L-homocysteine), and E (γ -glutamyl-methylseleno cysteine) for 6 hours (150ppb each compound). (A) Western blot photograph. (B-C) quantitative analysis of (B) phosphorylated FOXO3 at T32 and (C) phosphorylated FOXO4 at T28 in AML-12 cells shown in the above Western blot. Data are expressed as the mean of 3 samples + sem. The different letters (a and b) in the histogram mean that there are significant differences between the two groups: (P < 0.05)。

Figure 16 shows western blot analysis of various listed molecules in mouse hepatic AML-12 cells after treatment with vehicle (water, control) or 150ppb each of compound C (5' -methylselenoadenosine), D (Se-adenosyl-L-homocysteine) or E (γ -glutamyl-methylseleno-cysteine) for 6 and 24 hours.

Figure 17 shows that glucose 6 phosphatase catalytic subunit ("G6 pc") expression in mouse liver AML-12 cells was significantly down-regulated by a combination of compounds C (5' -methylselenoadenosine), D (Se-adenosyl-L-homocysteine), and E (γ -glutamyl-methylselenocysteine), but not by the individual compounds. Cells were treated with vehicle (control), selenium compound alone (150 parts per billion (150ppb)) or CDE combination (150ppb each) and their respective sulfur analogs for 48 hours, followed by quantitative RT-PCR. Data are presented as mean + sem of 4 samples in the histogram.

Figure 18 shows a schematic representation of the combination of compound CDE modulating G6pc expression in hepatocytes.

FIG. 19 shows the results of the analysis of the human Gsk3b, PSEN andNICASTRINthe presence of the FOXO binding motif in the promoter region of the gene, and a schematic of the effect of compound C on the regulation of important mediators in plaque and tangle formation in neuronal cells. Panel (A) shows in humansGSK3B5 FOXO1/3/4 binding motif positions on the promoter. Panel (B) shows in humansPSENThe position of two FOXO1/3/4 binding motifs on the promoter. Panel (C) shows in humansNicastrin (NCSTN)The position of the FOXO1/3/4 binding motif on the promoter. Panel (D) shows a schematic of the effect of compound C on the modulation of important mediators in the plaque and tangle formation of neuronal cells.

Detailed Description

Definition of

The terms "administration" and "administering" as used herein refer to the act of administering a drug, prodrug or other agent or therapeutic treatment (e.g., a composition of the present application) to a subject (e.g., a subject or in vivo, in vitro or ex vivo cells, tissues and organs). Exemplary routes of administration to the human body can be through the eye (ocularly), mouth (orally), skin (topical or transdermal), nose (nasally), lungs (inhalations), oral mucosa (buccally), ear, rectum, vagina, by injection (e.g., intravenously, subcutaneously, intratumorally, intraperitoneally, etc.), and the like.

The term "alkyl" refers to a branched or unbranched saturated hydrocarbon group of 1-24 carbon atoms. Preferred "alkyl" groups herein contain 1 to 16 carbon atoms; i.e. C_1-16An alkyl group. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentylHexyl, isohexyl, 3-methylpentyl, 2, 3-dimethylbutyl, neohexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl and hexadecyl. Most preferred is "lower alkyl", which refers to an alkyl group of 1 to 6, more preferably 1 to 4 carbon atoms. The alkyl group may be optionally substituted with an acyl group, amino group, amide group, azide group, carboxyl group, alkyl group, aryl group, halo group, guanidino group, oxo group, sulfanyl group, sulfoxy group, sulfonyl group, heterocyclic group, or hydroxyl group.

The term "alkali metal" refers to metal salts, including but not limited to suitable alkali metal (group la) salts, alkaline earth metal (group Ila) salts, and other physiologically acceptable metals. Such salts can be prepared from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc.

The term "alkenyl" refers to a straight or branched carbon chain containing at least 1 carbon-carbon double bond. In exemplary embodiments, "alkenyl" refers to hydrocarbons containing 2,3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms, i.e., C _1-10An alkenyl group. Examples of alkenyl groups include, but are not limited to, ethylene, propylene, butene, pentene, hexene, heptene, octene, nonene, and decene. Alkenyl groups may be optionally substituted with amino, alkyl, halo or hydroxy.

The term "amido" refers to a C-amido group such as — CONR ' R "or N-amido group such as — NR ' COR", where R ' and R "used in this definition are independently hydrogen, alkyl, alkenyl, alkynyl, alkoxy, carbocyclic, heterocyclic, aryl, or aralkyl. "sulfonamido" includes- -NR' - - -SO₂- - -R ''. Most preferably, R ' and R ' ' are hydrogen, alkyl, aryl or aralkyl.

The term "alkynyl" refers to a straight or branched carbon chain containing at least 1 carbon-carbon triple bond. In exemplary embodiments, "alkynyl" refers to a hydrocarbon containing 2,3,4, 5, 6, 7, 8, 9, or 10 carbon atoms, i.e., C_2-10Alkynyl. Examples of alkynyl groups include, but are not limited to, acetylene, propyne, butyne, pentyne, hexyne, heptyne, octyne, nonyne, and decyne. Alkynyl groups may be optionally substituted with amino, alkyl, halo or hydroxy.

The term "aryl" refers to a carbocyclic aromatic system containing 1,2, or 3 rings, wherein the rings may be linked together in a pendant manner, or may be fused. The term "fused" means that the second ring is present (i.e., connected or formed) by having two adjacent atoms that are common (i.e., shared) with the first ring. The term "fused" is equivalent to the term "condensed". The term "aryl" encompasses aromatic groups such as phenyl, naphthyl, tetrahydronaphthyl, 1,2,3, 4-tetrahydronaphthalene, indane, indene, and biphenyl. Aryl groups may be optionally substituted with amino, alkyl, halo, hydroxy, carbocyclic, heterocyclic, or another aryl group.

The term "cycloalkyl" refers to a monocyclic saturated or partially saturated carbocyclic ring in which the number of ring atoms is indicated by a numerical range. In exemplary embodiments, "cycloalkyl" refers to a carbocyclic ring containing 3 to 12 ring atoms as defined above (i.e., C)_3-12Cycloalkyl groups). As used herein, cycloalkyl includes monocyclic, bridged, spiro, fused, bicyclic, and tricyclic ring structures. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, norbornyl, decalin, adamantyl, and cyclooctyl. Cycloalkyl groups may be optionally substituted with amino, alkyl, halo or hydroxy.

The term "aralkyl" refers to an aryl-substituted alkyl moiety. Preferred aralkyl groups are "lower aralkyl" groups having an aryl group attached to an alkyl group of 1 to 6 carbon atoms. Examples of such groups include benzyl, benzhydryl, trityl, phenylethyl and diphenylethyl. The terms benzyl and phenylmethyl are used interchangeably.

The term "aryloxy" refers to an aryl group as defined above attached to an oxygen atom. Aryloxy may be optionally substituted with halo, hydroxy or alkyl. Examples of such a group include phenoxy group, 4-chloro-3-ethylphenoxy group, 4-chloro-3-methylphenoxy group, 3-chloro-4-ethylphenoxy group, 3, 4-dichlorophenoxy group, 4-methylphenoxy group, 3-trifluoromethoxy phenoxy group, 3-trifluoromethylphenoxy group, 4-fluorophenoxy group, 3, 4-dimethylphenoxy group, 5-bromo-2-fluorophenoxy group, 4-bromo-3-fluorophenoxy group, 4-fluoro-3-methylphenoxy group, 5,6,7, 8-tetrahydronaphthyloxy group, 3-isopropylphenoxy group, 3-cyclopropylphenoxy group, 3-ethylphenoxy group, 4-tert-butylphenoxy group, 3-pentafluoroethylphenoxy group and 3- (1,1,2, 2-tetrafluoroethoxy) phenoxy.

The term "alkoxy" refers to an oxy-containing group substituted with an alkyl or cycloalkyl group. Examples include, but are not limited to, methoxy, ethoxy, t-butoxy, and cyclohexyloxy. Most preferred are "lower alkoxy" groups having 1 to 6 carbon atoms. Examples of such groups include methoxy, ethoxy, propoxy, butoxy, isopropoxy, and tert-butoxy.

The term "aralkoxy" refers to an aralkyl group containing an oxy group attached through an oxygen atom to another group. "lower aralkyloxy" are those phenyl groups attached to a lower alkoxy group as described above. Examples of such groups include benzyloxy, 1-phenylethoxy, 3-trifluoromethoxy-benzyloxy, 3-trifluoromethyl-benzyloxy, 3, 5-difluorobenzyloxy, 3-bromobenzyloxy, 4-propylbenzyloxy, 2-fluoro-3-trifluoromethyl-benzyloxy and 2-phenylethoxy.

The term "acyl" refers to-C (= O) R, where R used in this definition is hydrogen, alkyl, alkenyl, alkynyl, carbocycle, heterocycle, aryl, or aralkyl. Most preferably, R is hydrogen, alkyl, aryl or aralkyl.

The term "carboxy" refers to — R ' C (= O) OR ", where R ' and R" used in this definition are independently hydrogen, alkyl, alkenyl, alkynyl, carbocycle, heterocycle, heterocycloalkyl, aryl, ether, OR aralkyl, OR R ' may additionally be a covalent bond. "carboxy" includes both carboxylic acids and carboxylic acid esters. The term "carboxylic acid" refers to a carboxyl group wherein R "is hydrogen or a salt. Such acids include formic acid, acetic acid, propionic acid, butyric acid, valeric acid, 2-methylpropionic acid, ethylene oxide-formic acid and cyclopropanecarboxylic acid. The term "carboxylate" or "ester" refers to a carboxyl group wherein R "is alkyl, alkenyl, alkynyl, carbocycle, heterocycle, aryl, or aralkyl. Examples of carboxylic acids include, but are not limited to, formic acid, acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, enanthic acid, caprylic acid, pelargonic acid, capric acid, cyclopropanecarboxylic acid, cyclobutanecarboxylic acid, cyclopentanecarboxylic acid, cyclohexanecarboxylic acid, cycloheptanecarboxylic acid, cyclooctanecarboxylic acid, or cyclononanecarboxylic acid.

The term "carbonyl" refers to a C = O moiety, also known as an "oxo" group.

The term "heterocycle" or "heterocyclyl" or "heterocyclic ring" refers to an optionally substituted, saturated or unsaturated, aromatic or non-aromatic cyclic hydrocarbon having 3 to 12 or 5 to 6 carbon atoms, wherein at least 1 ring atom is O, N, S, P or Se. For example, in some embodiments, the ring N atom from the heterocyclyl group is a bonding atom to form an amide, carbamate, or urea with-c (o). In exemplary embodiments, "heterocyclyl" refers to the above-described cyclic hydrocarbons containing 4, 5, or 6 ring atoms (i.e., C)_4-6A heterocyclic group). Examples of heterocyclyl groups include, but are not limited to, aziridine, oxirane, thietane, azetidine, epoxypropane, thietane, pyrrolidine, imidazole, tetrahydrofuran, pyran, thiopyran, thiomorpholine S-oxide, oxazoline, tetrahydrothiophene, piperidine, tetrahydropyran, thiacyclohexane (thiane), imidazolidine, oxodioxolyl, oxazolidine, thiazolidine, dioxolane, dithiolane, piperazine, oxazine, dithiane, dioxane, pyridyl, furyl, benzofuryl, isobenzofuryl, pyrrolyl, thienyl, 1,2, 3-triazolyl, 1,2, 4-triazolyl, indolyl, imidazolyl, thiazolyl, thiadiazolyl, pyrimidinyl, oxazolyl, triazinyl, and tetrazolyl. Exemplary heterocycles include benzimidazole, dihydrothiophene, dioxane, dioxolane, dithiane, dithiazole, dithiacyclopentane, furan, indole, 3-H indazole, 3-H-indole, indolizine, isoindole, isothiazole, isoxazole, morpholine, oxazole, oxadiazole, oxathiazole, oxathiazolidine, oxazine, oxadiazine, piperazine, piperidine, purine, pyran, pyrazine, pyrazole, pyridine, pyrimidine, pyridazine, pyrrole, pyrrolidine, tetrahydrofuran, tetrazine, thiadiazine, thiadiazole, thiatriazole, thiazine, thiazole, thiophene, triazine, and triazole. The heterocycle may be optionally substituted with amino, alkyl, alkenyl, alkynyl, halo, hydroxy, carbocycle, thio, other heterocycle, or aryl.

The term "heteroaryl" refers to a cyclic hydrocarbon in which at least 1 ring atom is O, N, S, P or Se, the ring being characterized by a delocalization [ pi ] shared among the ring members]Electron (aromatic), and wherein the ring atomsNumbers are indicated by a range of values. Heteroaryl moieties as defined herein have C, N, S, P or a pendant Se bonding pendant group (bonding hands). For example, in some embodiments, the ring N atom from the heteroaryl group is a bonding atom to form an amide, carbamate, or urea with-c (o). In exemplary embodiments, "heteroaryl" refers to the above-described cyclic hydrocarbons containing 5 or 6 ring atoms (i.e., C)_5-6Heteroaryl). Examples of heteroaryl groups include, but are not limited to, pyrrole, furan, thiophene, oxazole, thiazole, isoxazole, isothiazole, imidazole, pyrazole, oxadiazole, thiadiazole, triazole, tetrazole, pyridine, pyrimidine, pyrazine, pyridazine, and triazine.

The term "hydroxy" or "hydroxy" refers to the substituent-OH.

The term "oxo" refers to substituent = O.

The term "nitro" means NO₂。

The term "azido" refers to N₃。

The term "sulfur analog" refers to an analog of a compound of interest in which 1 or more selenium atoms are replaced by 1 or more sulfur atoms, respectively.

The term "thioalkyl" refers to- -SR ', where R' used in this definition is hydrogen, alkyl, alkenyl, alkynyl, carbocycle, heterocycle, aryl, or aralkyl.

The term "thionylene" refers to — SOR ', where R' used in this definition is hydrogen, alkyl, alkenyl, alkynyl, carbocycle, heterocycle, aryl, or aralkyl.

The term "sulfonyl" refers to — SOR ', where R' refers to hydrogen, alkyl, alkenyl, alkynyl, carbocycle, heterocycle, aryl, or aralkyl.

The term "ketone" refers to a moiety containing at least 1 carbonyl group, wherein the carbonyl carbon is bonded to two other carbon atoms. In exemplary embodiments, "ketone" refers to a carbonyl-containing moiety (i.e., C) described above containing 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms_3-10Ketones). Examples of ketone groups include, but are not limited to, acetone, butanone, pentanone, hexanone, heptanone, octanone, nonanone, decanone, cyclobutanone, cyclopentanone, cyclohexanone, cycloheptanone, cyclooctanone, cyclononanone, and cyclodecanone.

The term "amino" refers to a primary, secondary or tertiary amino group of the formula — NR ' R ", where R ' and R" used in this definition are independently hydrogen, acyl, alkyl, alkenyl, alkynyl, aralkyl, aryl, carboxyl, cycloalkyl, heterocyclic or other amino groups (in the case of hydrazides) or R ' and R "together with the nitrogen atom to which they are attached form a ring having 4 to 8 atoms. Thus, the term "amino" includes both unsubstituted, monosubstituted (e.g., monoalkylamino or monoarylamino) and disubstituted (e.g., dialkylamino or aralkylamino) amino groups. Amino radicals comprising- -NH ₂Methylamino, ethylamino, dimethylamino, diethylamino, methyl-ethylamino, pyrrolidin-1-yl, piperidino, morpholino, and the like. Other exemplary ring-forming "amino" groups include pyrrolyl, imidazolyl, pyrazolyl, isothiazolyl, isoxazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolizinyl, imidazolyl, isoindolyl, indolyl, indazolyl, purinyl, quinolizinyl. The amino-containing ring may be optionally substituted with another amino, alkyl, alkenyl, alkynyl, halo, or hydroxy group.

The term "amine" refers to a primary, secondary or tertiary amino group of the formula — NR ' R ", where R ' and R" used in this definition are independently hydrogen, acyl, alkyl, alkenyl, alkynyl, aralkyl, aryl, carboxyl, cycloalkyl, heterocyclic or other amino groups (in the case of hydrazides) or R ' and R "together with the nitrogen atom to which they are attached form a ring having 4 to 8 atoms. Thus, the term "amino" as used herein includes both unsubstituted, monosubstituted (e.g., monoalkylamino or monoarylamino) and disubstituted (e.g., dialkylamino or aralkylamino) amino groups. Amino radicals comprising- -NH₂Methylamino, ethylamino, dimethylamino, diethylamino, methyl-ethylamino, pyrrolidin-1-yl, piperidino, morpholino, and the like. Other exemplary ring-forming "amino" groups include pyrrolyl, imidazolyl, pyrazolyl, isothiazolyl, isoxazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolizinyl, imidazolyl, isoindolyl, indolyl, indazolyl, purinyl, quinolizinyl. The amino-containing ring may be optionally substituted with another amino, alkyl, alkenyl, alkynyl, halo, or hydroxy group.

The term "alcohol" means "hydroxy" or "hydroxy" means the substituent-OH.

The term "aminoalcohol" refers to a functional group that contains both an alcohol and an amine group. As used herein, "amino alcohol" also refers to an amino acid as defined above having a carbon bonded to the alcohol in place of the carboxylic acid group. In exemplary embodiments, the term "aminoalcohol" refers to an aminoalcohol as defined above in which an amine is bonded to a carbon adjacent to the carbon bearing the alcohol. In exemplary embodiments, "aminoalcohol" refers to an amine and alcohol containing moiety (i.e., C) as defined above containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms (i.e., C)_1-12Amino alcohol). Examples of aminoalcohols include, but are not limited to, ethanolamine, heptamine, ethyl isoproterenol, norepinephrine, propanolamine, sphingosine, methanolamine, 2-amino-4-mercaptobutan-1-ol, 2-amino-4- (methylthio) butan-1-ol, cysteine, phenylglycinol, prolinol, 2-amino-3-phenyl-1-propanol, 2-amino-l-propanol, cyclohexylglycinol, 4-hydroxy-prolinol, leucinol, tert-leucinol, phenylalaninol, a-phenylglycinol, 2-pyrrolidinemethanol, tyrosinol, valinol, serinol, 2-dimethylaminoethanol, histidinol, isoleucinol, leucinol, methioninol, l-methyl-2-pyrrolidinemethanol, threoninol, tryptophanol, alaninol, argininol, glycinol, glutaminol, 4-amino-5-hydroxypentanamide, 4-amino-5-hydroxypentanoic acid, 3-amino-4-hydroxybutyric acid, lysinol, 3-amino-4-hydroxybutyramide and 4-hydroxy-prolinol.

The term "amino acid" refers to a group containing a carboxylic acid and an amine bound to the carbon atom immediately adjacent to the carboxylic acid group, and includes both natural and synthetic amino acids. Examples of amino acids include, but are not limited to, arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, selenocysteine, glycine, proline, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, tryptophan. The carboxyl group is substituted with H, a salt, an ester, an alkyl group or an aralkyl group. The amino group is substituted with H, acyl, alkyl, alkenyl, alkynyl, carboxyl, cycloalkyl, aralkyl, or heterocyclic group.

The term "ether" refers to the group-R ' - -O-R ", wherein R ' and R" used in this definition are independently hydrogen, alkyl, alkenyl, alkynyl, carbocycle, heterocycle, aryl, or aralkyl, and R ' may additionally be a covalent bond attached to carbon.

The term "halogen" refers to a fluorine, chlorine, bromine or iodine atom.

The term "halide" refers to a functional group containing an atom bonded to a fluorine, chlorine, bromine, or iodine atom. Exemplary embodiments disclosed herein may include "alkyl halide", "alkenyl halide", "alkynyl halide", "cycloalkyl halide", "heterocyclyl halide", or "heteroaryl halide" groups. In exemplary embodiments, "alkyl halide" refers to a moiety containing a carbon-halogen bond that contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms (i.e., C) _1-10Alkyl halides). Examples of alkyl halide groups include, but are not limited to, fluoromethyl, fluoroethyl, chloromethyl, chloroethyl, bromomethyl, bromoethyl, iodomethyl, and iodoethyl. Unless otherwise specified, any carbon-containing group referred to herein may contain 1 or more carbon-halogen bonds. By way of non-limiting example, Ci alkyl may be, but is not limited to, methyl, fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, bromomethyl, dibromomethyl, tribromomethyl, iodomethyl, diiodomethyl, triiodomethyl, chlorofluoromethyl, dichlorofluoromethyl, and difluorochloromethyl.

In the compounds described herein, the heteroatoms can have a plurality of different valences. By way of non-limiting example, S, Se and N may be neutral or positively charged, and O may be neutral or positively or negatively charged.

In some embodiments, the compounds of formula (I), (II), or (III) include diastereomers and enantiomers of the indicated compounds. An enantiomer is defined as one of a pair of molecular entities that are mirror images and non-overlapping with each other. Diastereomers or diastereomers are defined as stereoisomers of diastereomers. Diastereomers or diastereomers are stereoisomers that do not relate as mirror images. Diastereomers are characterized by differences in physical properties.

The term "compound C" or "compound C" as shown herein refers to 5' -methylselenoadenosine; also known as (2R,4S,5S) -2- (6-amino-9H-purin-9-yl) -5- ((methylselenoalkyl) methyl) tetrahydrofuran-3, 4-diol, CAS registry number 5135-40-0, and including any pharmaceutically acceptable salts thereof.

The term "compound D" or "compound D" as shown herein refers to 5' -selenoadenosine homocysteine; (2R) -2-amino-4- (((((2S, 3S,5R) -5- (6-amino-9H-purin-9-yl) -3, 4-dihydroxytetrahydrofuran-2-yl) methyl) selenoalkyl) butanoic acid, CAS registry No. 4053-91-2, and including any pharmaceutically acceptable salts thereof.

The term "compound E" or "compound E" as shown herein refers to γ -glutamyl-methylseleno-cysteine or γ -L-glutamyl-Se-methyl-L-cysteine; also known as N5- (1-carboxy-2- (methylselenoalkyl) ethyl) -L-glutamine or any pharmaceutically acceptable salt thereof.

The term "compound H" or "compound H" as indicated herein refers to 5' -methylthioadenosine; 5 '-S-methyl-5' -thioadenosine, CAS registry No. 2457-80-9, or a pharmaceutically acceptable salt thereof.

The term "compound I" or "compound I" refers to S-adenosyl-L-homocysteine, also known as (S) -5'- (S) - (3-amino-3-carboxypropyl) -5' -thioadenosine, CAS registry No. 979-92-0, or a pharmaceutically acceptable salt thereof.

The term "compound J" or "compound J" as used herein refers to γ -L-glutamyl-methyl-L-cysteine, also known as γ -glutamyl-methyl-cysteine or a pharmaceutically acceptable salt thereof.

The term "compound CDE" refers to a mixture of compound C, compound D, and compound E, or a pharmaceutically acceptable salt thereof.

The term "compound HIJ" refers to a mixture of compound H, compound I, and compound J, or a pharmaceutically acceptable salt thereof.

The terms "analog" and "derivative" are used interchangeably and refer to a natural or unnatural modification of at least 1 position of a given molecule. For example, derivatives of a given compound or molecule are modified by the addition of functional groups or atoms, the removal of functional groups or atoms, or the changing of functional groups or atoms to different functional groups or atoms (including but not limited to isotopes).

The term "comprising" means a composition, compound, formulation or method that includes and does not exclude other elements or method steps.

The term "consisting of … …" refers to a compound, composition, formulation, or method that excludes the presence of any other component or method step.

The term "consisting essentially of … …" refers to a composition, compound, formulation or method that includes additional elements or method steps that do not materially affect the characteristics of the composition, compound, formulation or method.

The term "compound" refers to any one or more chemical entities, agents, drugs, etc., that can be used to treat or prevent a disease, illness, discomfort or condition of bodily function. The compounds include both known and potential therapeutic compounds. A compound can be determined to be therapeutic by screening using the screening methods of the present application. By "known therapeutic compound" is meant a therapeutic compound that has been shown (e.g., by animal testing or prior experimentation administered to a human) to be effective in the treatment. In other words, known therapeutic compounds are not limited to compounds that are effective in treating diseases (e.g., neurodegenerative diseases).

The term "composition" refers to a combination of one or more compounds with or without another agent, such as, but not limited to, a carrier agent (e.g., one or more selenium-containing compounds with an inert or active carrier, such that the composition is particularly suitable for diagnostic or therapeutic use in vitro, in vivo, or ex vivo).

The term "component" refers to a compound or constituent of a composition. For example, a component of a composition can include a compound, a carrier, and any other agents present in the composition.

The term "effective amount" refers to an amount of a composition or compound sufficient to achieve a beneficial or desired result. An effective amount may be administered in one or more administrations or dosages, and is not intended to be limited to a specific formulation or route of administration.

The term "hydrate" refers to a compound disclosed herein that is molecularly associated with water, i.e., wherein the H — OH bond is unbroken, and can be represented, for example, by the formula rxh₂O, wherein R is a compound disclosed herein. A given compound may form more than one hydrate, including, for example, monohydrate (R x H)₂O), dihydrate (R)₂ x H₂O), trihydrate (R)₃ x H₂O), and the like.

The term "inhibitory" or "antagonistic" refers to the property of a compound that reduces, limits, or blocks the action or function of another compound.

The term "isolated" refers to a material in a mixture that is separated from at least one other material, or from a material with which the material naturally accompanies. For example, the synthesized compounds are isolated from starting materials or intermediates.

The term "mitochondrial potential" refers to the voltage difference across the inner mitochondrial membrane, which is maintained by the net positive charge movement across the membrane.

The term "modulate" refers to a change in the state (e.g., activity or amount) of a compound from a known or determined state.

"optional" or "optionally" refers to situations in which the subsequently described event or circumstance may or may not occur, and includes instances where said event or circumstance occurs and instances where it does not. "optionally" includes embodiments wherein said conditions are present and embodiments wherein said conditions are absent. For example, "optionally substituted phenyl" means that the phenyl group may or may not be substituted, and the description includes both unsubstituted phenyl and phenyl groups in which substitution is present. "optionally" includes embodiments wherein said conditions are present and embodiments wherein said conditions are absent.

The term "organoselenium" or "seleno-organic compound" refers to any organic compound in which selenium replaces sulfur. Thus, organoselenium may refer to any such compound biosynthesized by yeast, or it may refer to a chemically synthesized free organoselenium compound. An example of the latter is free selenomethionine. In some cases, the selenium organic compound also includes a selenium metabolite.

The terms "patient" or "subject" are used interchangeably and refer to any member of the kingdom animalia. Preferably the subject is a mammal, for example a human, domestic mammal or livestock mammal.

The phrase "pharmaceutically acceptable" refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The phrase "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the selenium-containing compound or analog or derivative from one organ or portion of the body to another organ or portion of the body. The various carriers must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials that can be used 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) tragacanth powder; (5) malt; (6) gelatin; (7) talc powder; (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 glycerol, 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 hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) a ringer's solution; (19) ethanol; (20) a phosphate buffer solution; and (21) other non-toxic compatible materials used in pharmaceutical formulations.

The term "prodrug" refers to a pharmacologically active or more generally inactive compound that is converted to a pharmacologically active agent by metabolic conversion. Prodrugs of compounds of any of the above formulae are prepared by modifying functional groups present in compounds of any of the above formulae in such a way that the modifications are cleavable in vivo to release the parent compound. In vivo, prodrugs readily undergo chemical changes (e.g., hydrolysis or exposure to naturally occurring enzymes) under physiological conditions, resulting in the release of the pharmacologically active agent. Prodrugs include compounds of any of the above formulas wherein a hydroxy, amino, or carboxyl group is bonded to any group that can be cleaved in vivo to regenerate the free hydroxy, amino, or carboxyl group, respectively. Examples of prodrugs include, but are not limited to, esters (e.g., acetate, formate, and benzoate derivatives) of any of the above compounds or any other derivative that converts to the active parent drug at physiological pH or by the action of enzymes. Conventional procedures for selecting and preparing suitable prodrug derivatives are described in the art (see, e.g., bundgaard. Design of produgs. Elsevier, 1985).

The term "purified" or "purification" or "substantially purified" refers to the removal of a component, e.g., an inactive, inhibitory component, unreacted compound, or replacement compound (e.g., contaminant) produced in a synthesis, from a composition to the extent that 10% or less (e.g., 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less) of the composition is not an active compound or pharmaceutically acceptable carrier.

The term "salt" may include acid addition salts or addition salts of the free base. Preferably the salt is pharmaceutically acceptable. Examples of acids that can be used to form pharmaceutically acceptable acid addition salts include, but are not limited to, salts derived from non-toxic inorganic acids such as nitric, phosphoric, sulfuric, or hydrobromic, hydroiodic, hydrofluoric, phosphorous, and salts derived from non-toxic organic acids such as aliphatic mono-and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, alkanedioic acids, aromatic acids, aliphatic and aromatic sulfonic and acetic acids, maleic, succinic, or citric acids. Non-limiting examples of such salts include naphthalene disulfonates, benzene sulfonates, sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphates, hydrochlorides, hydrobromides, hydroiodides, acetates, trifluoroacetates, propionates, octanoates, isobutyrates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, mandelates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, phthalates, benzenesulfonates, tosylates, phenylacetates, citrates, lactates, maleates, tartrates, methanesulfonates, and the like. Salts of amino acids such as arginine Salts and the like and gluconate, galacturonate (see, e.g., Berge, et al, "Pharmaceutical Salts,") " J. Pharma. Sci. 1977;66:1)。

The term "pharmaceutically acceptable salts" includes, but is not limited to, salts well known to those skilled in the art, such as mono (e.g., alkali metal and ammonium) and poly (e.g., di-or tri-salts) salts of the compounds of the present invention. Pharmaceutically acceptable salts of the compounds of the present disclosure are those in which, for example, the hydrogen in an exchangeable group, e.g., -OH, - — NH- -or-P (= O) (OH) —, is replaced with a pharmaceutically acceptable cation (e.g., sodium, potassium or ammonium ion), and may be suitably prepared from the corresponding compounds disclosed herein, e.g., by reaction with a suitable base. Where the compound is sufficiently basic or acidic to form a stable, non-toxic acid or base salt, administration of the compound as a salt may be suitable. Examples of pharmaceutically acceptable salts are organic acid addition salts with acids forming physiologically acceptable anions, such as tosylate, mesylate, acetate, citrate, malonate, tartrate, succinate, benzoate, ascorbate, alpha-ketoglutarate and alpha-glycerophosphate. Suitable inorganic salts may also be formed, including hydrochlorides, sulfates, nitrates, bicarbonates, and carbonates. Pharmaceutically acceptable salts can be obtained using standard procedures well known in the art, for example by reacting a sufficiently basic compound such as an amine with a suitable acid to provide a physiologically acceptable anion. Alkali metal (e.g., sodium, potassium, or lithium) or alkaline earth metal (e.g., calcium) salts of carboxylic acids may also be prepared.

The terms "selenium-enriched yeast" and "selenized yeast" refer to any yeast (e.g., saccharomyces cerevisiae) cultured in a medium containing an inorganic selenium salt. The application is not limited to the selenium salt used. Indeed, it is contemplated that various selenium salts may be used in the present application, including but not limited to sodium selenite, sodium selenate, cobalt selenite, or cobalt selenate. The selenium-containing compound in such yeast preparations is selenomethionine, which is present in a form incorporated into the polypeptide/protein. The amount of total cellular selenium present in the form of selenomethionine in such preparations will vary, but can be between 10 and 100%, 20-60%, 50-75% and between 60 and 75%. The remainder of the organic selenium in the selenized yeast preparation is mainly composed of intermediates in the selenomethionine biosynthetic pathway. These include, but are not limited to, selenocysteine, selenohomocysteine, and selenadenosylselenomethionine. The amount of residual inorganic selenium salt in the final product is typically very low (e.g. < 2%).

The term "substituted" with respect to a moiety (moiey) refers to an additional substituent attached to the moiety at any acceptable position on the moiety. Unless otherwise indicated, moieties may be bonded through carbon, nitrogen, oxygen, sulfur, or any other acceptable atom. Examples of substituents include, but are not limited to, amines, alcohols, thiols, ethers, alkenes, alkynes, epoxides, aziridines, oxiranes, azetidines, dihydrofurans, pyrrolidines, pyrans, piperidines, aldehydes, ketones, esters, carboxylic acids, carboxylic esters, imines, imides, azides, azo groups, enamines, alkyl halides, alkenyl halides, alkynyl halides, aryl halides, phosphines, phosphine oxides, phosphinates, phosphonites, phosphites, phosphonates, phosphates, sulfates, sulfoxides, sulfonyl groups, sulfoxyl (sulfoxyl), sulfonates, nitrates, nitrites, nitriles, nitro groups, nitroso groups, cyanates, thiocyanates, isothiocyanates, carbonates, acyl halides, peroxides, hydroperoxides, hemiacetals, hemiketals, acetals, orthoesters, orthocarbonates, sulfides, disulfides, ketals, orthoesters, carbonates, dithiols, aryl halides, phosphine, and the like, Sulfonic acids, thioketones, thioaldehydes, phosphodiesters, boric acids, boric acid esters, boric acids, and boric acid esters.

The term "treatment" refers to a therapeutic treatment in which the objective is to slow down (e.g., reduce or delay the onset of) an undesired physiological condition, disorder or disease, or to achieve a beneficial or desired result, such as partial or total restoration or inhibition of a decline in a parameter, value, function or result that has or will become abnormal. Beneficial or desired results include, but are not limited to, alleviation of symptoms; a reduction in the extent or viability or rate of progression of a condition, disorder or disease; stabilization (i.e., not worsening) of the state of the condition, disorder or disease; delayed onset or slowed progression of the condition, disorder or disease; amelioration of a condition, disorder or disease state; and reduction (whether partial or total) of actual clinical symptoms (whether or not it translates into a direct reduction of actual clinical symptoms) or an increase or amelioration of the condition, disorder or disease.

The term "agent capable of specifically detecting gene expression" refers to an agent capable of or sufficient to detect the expression of the various genes described in detail herein. Examples of suitable reagents include, but are not limited to, nucleic acid probes capable of specifically hybridizing to mRNA or cDNA, and antibodies (e.g., monoclonal or polyclonal antibodies).

The term "toxic" refers to any deleterious or detrimental effect on a subject, cell or tissue as compared to the same cell or tissue prior to administration of a toxic agent.

Compounds and compositions

One aspect of the present application relates to 5' -methylselenoadenosine ("compound C"), Se-adenosyl-L-homocysteine ("compound D"), gamma-glutamyl-methylseleno-cysteine ("compound E"), and analogs thereof. Some embodiments include compositions comprising compounds of formula I, formula II, and/or formula III, and combinations thereof.

In some embodiments, there is provided a pharmaceutical composition comprising a compound of formula (I):

or a pharmaceutically acceptable salt, hydrate or prodrug thereof,

wherein R is₁Is H, acyl, alkyl, alkenyl, alkynyl, aralkyl, carboxyl, cycloalkyl, C (O) R ', C (O) OR ', wherein R ' is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, OR heterocyclyl; or R₁And R₂Together form a heterocyclic ring having 4-8 ring members and at least one heteroatom selected from oxygen or nitrogen;

R₂is H, acyl, alkyl, alkenyl, alkynyl, aralkyl, carboxy, cycloalkyl, C (O) R ', C (O) OR ', wherein R ' is selected from alkyl, cycloalkyl, aryl, aralkyl, OR heterocyclyl; or R ₁And R₂Together form a heterocyclic ring having 4-8 ring members and at least one heteroatom selected from oxygen or nitrogen;

R₄is H, acyl, alkylA group, alkenyl, alkynyl, aralkyl, cycloalkyl, carboxyl or C-amido; or R₃And R₄Together with the atoms to which they are attached form a heterocyclic ring having 4-8 ring members and at least one heteroatom selected from oxygen or nitrogen;

R₅is oxo, hydroxy, alkyl, alkenyl, alkynyl, OR' OR absent; wherein R' is selected from alkyl, alkenyl, alkynyl, cycloalkyl, aryl or aralkyl;

R₆is oxo, hydroxy, alkyl, alkenyl, alkynyl, OR' OR absent; wherein R' is selected from alkyl, alkenyl, alkynyl, cycloalkyl, aryl or aralkyl;

R₇is H, alkyl, alkenyl, alkynyl, ketone, amino alcohol, amino acid, OR ', Se-R', S-R ', wherein R' is selected from H, alkyl, cycloalkyl, aryl, aralkyl, OR heterocyclyl; and

R₈is hydrogen, azido, alkyl, alkenyl, alkynyl.

In a further embodiment, one or more of these compounds of formula (I) may be isolated and/or purified.

In some embodiments, there is provided a composition comprising a compound of formula (I), or a pharmaceutically acceptable salt, hydrate, or prodrug thereof, wherein R is₁、R₃、R₄And R₈Each is H; r₂Is H, acyl, alkyl, alkenyl, alkynyl, aralkyl, carboxy, cycloalkyl, C (O) R ', C (O) OR ', wherein R ' is selected from alkyl, cycloalkyl, aryl, aralkyl, OR heterocyclyl; r₅And R₆Each is absent; and R₇Is an alkyl or amino acid(ii) a Provided that 5 '-selenoadenosine methionine, dehydroxy 5' -methylselenoadenosine, ethylselenoadenosine, seleno (hydroxy) -selenophenyl- (3 '-deoxy-adenosine), allylselenoadenosine homocysteine, selenoadenosine-Se (methyl) -selenoxide, adenosine-hydroxyselenoxide, ethylselenoadenosine, seleno- (hydroxy) -selenophenyl- (3' -deoxy-adenosine), adenosine-hydroxyselenoxide, and selenoadenosine-Se (methyl) -selenoxide can each be excluded from the composition.

In a particular aspect, there is provided a composition comprising a compound of formula (I), or a pharmaceutically acceptable salt, hydrate or prodrug thereof, which compound is 5' -methylselenoadenosine ("compound C").

In some embodiments, the compositions comprise a compound of formula (I), or a pharmaceutically acceptable salt, hydrate, or prodrug thereof, provided that 5 '-selenoadenosine methionine, dehydroxy 5' -methylselenoadenosine, ethylselenoadenosine, seleno (hydroxy) -selenophenyl- (3 '-deoxy-adenosine), allylselenoadenosine homocysteine, selenoadenosine-Se (methyl) -selenoxide, adenosine-hydroxyselenoxide, ethylselenoadenosine, seleno- (hydroxy) -selenophenyl- (3' -deoxy-adenosine), adenosine-hydroxyselenoxide, and selenoadenosine-Se (methyl) -selenoxide can each be excluded from the composition.

In some embodiments, there is provided a composition comprising a compound of formula (II):

or a pharmaceutically acceptable salt, hydrate or prodrug thereof,

wherein R is₁Is H, acyl, alkyl, alkenyl, alkynyl, aralkyl, carboxyl, cycloalkyl, C (O) R ', C (O) OR ', wherein R ' is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, OR heterocyclyl; or R ₁And R₂Together form a ring having 4-8 ring members and being selected from oxygen or nitrogen toA heterocycle with one heteroatom less;

R₅is oxo, hydroxy, alkyl, alkenyl, alkynyl, OR' OR absent; wherein R' is selected from alkyl, alkenyl, alkynyl, cycloalkyl, aryl or aralkyl;

R₆is oxo, hydroxy, alkyl, alkenyl, alkynyl, OR' OR absent; wherein R' is selected from alkyl, alkenyl, alkynyl, cycloalkyl, aryl or aralkyl;

R₈Is hydrogen, azido, alkyl, alkenyl, alkynyl;

R₉is H, acyl, alkyl, alkenyl, alkynyl, aralkyl, carboxy, cycloalkyl, C (O) R ', C (O) OR ', wherein R ' is alkyl, cycloalkyl, aryl, aralkyl, OR heterocyclyl; or R₉And R₁₀Together form a heterocyclic ring having 4-8 ring members and at least one heteroatom selected from oxygen or nitrogen;

R₁₀is H, acyl, alkyl, alkenyl, alkynyl, aralkyl, carboxy, cycloalkyl, C (O) R ', C (O) OR ', wherein R ' is selected from alkyl, cycloalkyl, aryl, aralkyl, OR heterocyclyl; or R₉And R₁₀Together form a ring having 4-8 ring members and at least one member selected from oxygen or nitrogenA heterocyclic ring of a heteroatom; and

R₁₁is OH, OR, alkoxy, aralkoxy OR amino, wherein R is selected from alkyl, cycloalkyl, aryl, aralkyl, heterocyclyl OR a pharmaceutically acceptable salt OR inner salt.

In a further embodiment, one or more of these compounds of formula (II) may be isolated and/or purified.

In some embodiments, there is provided a composition comprising a compound of formula (II) or a pharmaceutically acceptable salt, hydrate, or prodrug thereof, wherein R is₁、R₃、R₄、R₈And R₉Each is H; r₂Is H, acyl, alkyl, carboxyl, C (O) R ' OR C (O) OR ', wherein R ' is selected from alkyl, cycloalkyl, aryl, aralkyl, OR heterocyclyl; r ₅And R₆Is absent; r₁₀Is H, acyl, alkyl, alkenyl, alkynyl, aralkyl, carboxy, cycloalkyl, C (O) R ', C (O) OR ', wherein R ' is selected from alkyl, cycloalkyl, aryl, aralkyl, OR heterocyclyl; and R₁₁Is OH, OR, alkoxy, aralkoxy OR amino, wherein R is selected from alkyl, cycloalkyl, aryl, aralkyl, heterocyclyl OR a pharmaceutically acceptable salt OR inner salt.

In some embodiments, there is provided a composition comprising a compound of formula (II) or a pharmaceutically acceptable salt, hydrate, or prodrug thereof, wherein R is₁、R₂、R₃、R₄、R₅、R₆、R₇、R₈、R₉、R₁₀As defined above and wherein R₁₁Is OH OR OR, wherein R is selected from methyl, ethyl, propyl, isopropyl, butyl, sec-butyl OR tert-butyl.

In a particular aspect, there is provided a composition comprising a compound of formula (II) or a pharmaceutically acceptable salt, hydrate or prodrug thereof, said compound being 5' -selenoadenosine homocysteine (compound "D") or a pharmaceutically acceptable salt, hydrate or prodrug thereof.

In some embodiments, the composition comprises a compound of formula (II) or a pharmaceutically acceptable salt, hydrate, or prodrug thereof; provided that 5' -selenoindenosine methionine, allylselenadenosylhomocysteine, selenadenosylhomocysteine and selenohydroxyadenosylhomocysteine can each be excluded from the composition.

In some embodiments, there is provided a pharmaceutical composition comprising a compound of formula (III):

or a pharmaceutically acceptable salt, hydrate or prodrug thereof, wherein

R₃is OH, OR, alkoxy, aralkoxy OR amino, wherein R is selected from alkyl, cycloalkyl, aryl, aralkyl, heterocyclyl OR a pharmaceutically acceptable salt OR inner salt;

R₄is H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocyclyl or a pharmaceutically acceptable salt or inner salt;

R₅is oxo, hydroxy, alkyl, alkenyl, alkynyl, OR' OR absent; wherein R' is selected from alkyl, alkenyl, alkynyl, cycloalkyl, aryl or aralkyl;

R₆Is oxo, hydroxy, alkyl, alkenyl, alkynyl, OR' OR absent; wherein R' is selected from alkyl, alkenyl, alkynyl, cycloalkyl, aryl or aralkyl; and

R₇is H, alkyl, alkenyl, alkynyl, ketoneOR ', Se-R', S-R ', wherein R' is selected from H, alkyl, cycloalkyl, aryl, aralkyl OR heterocyclic group.

In some embodiments, there is provided a composition comprising a compound of formula (III), or a pharmaceutically acceptable salt, hydrate, or prodrug thereof, wherein

R₁And R₂Each is H;

R₃is OH, OR, alkoxy, aralkoxy OR amino, wherein R is selected from alkyl, cycloalkyl, aryl, aralkyl, heterocyclyl OR a pharmaceutically acceptable salt OR inner salt;

R₄is H or a pharmaceutically acceptable salt or inner salt;

R₅and R₆Is absent; and

R₇is alkyl, alkenyl or alkynyl.

In a further embodiment, one or more of these compounds of formula (III) may be isolated and/or purified.

In some embodiments, there is provided a composition comprising a compound of formula (III), or a pharmaceutically acceptable salt, hydrate, or prodrug thereof, wherein R is₁And R₂Each is H; r₃Is OH OR OR, wherein R is selected from methyl, ethyl, propyl, isopropyl, butyl, sec-butyl OR tert-butyl; r ₄Is H; r₅And R₆Each is absent; and R₇Is an alkyl group which is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl or tert-butyl.

In some embodiments, there is provided a composition comprising a compound of formula III, or a pharmaceutically acceptable salt, hydrate, or prodrug thereof, wherein R is₁And R₂Each is H; r₃Is OH OR OR, wherein R is selected from methyl, ethyl, propyl, isopropyl, butyl, sec-butyl OR tert-butyl; r₄Is H; r₅And R₆Is absent; and R₇Is methyl.

In a particular aspect, there is provided a composition comprising a compound of formula (III), or a pharmaceutically acceptable salt, hydrate, or prodrug thereof, which compound is γ -glutamyl-methylseleno-cysteine ("compound E"), or a pharmaceutically acceptable salt, hydrate, or prodrug thereof.

In some embodiments, the composition comprises a compound of formula (III), or a pharmaceutically acceptable salt, hydrate, or prodrug thereof, provided that γ -glutamylselenocysteine- γ -glutamylcysteine, γ -glutamylcysteine-2, 3-DHP-selenocysteine, di- γ -glutamylselenocysteine, selenoglutazone- γ -glutamylcysteine, gamma-glutamylselenocysteine-gamma-glutamylcysteine, gamma-glutamylcysteine-2, 3-DHP-selenocysteine, di-gamma-glutamylselenocysteine and selenoglutazone-gamma-glutamylcysteine can each be excluded from the composition.

In some embodiments, compositions are provided comprising one or more compounds according to one or more of formulas (I), (II), and/or (III), wherein each of the following compounds is excluded from the composition to minimize selenium toxicity, remove inactive or inhibitory compounds, and/or maximize the therapeutic index of the composition, wherein the excluded compounds are γ -glutamylselenocysteine- γ -glutamylcysteine, γ -glutamylcysteine-2, 3-DHP-selenocysteine, di- γ -glutamylselenocysteine, selenoglutazone- γ -glutamylcysteine, γ -glutamylselenocysteine- γ -glutamylcysteine, γ -glutamylcysteine-2, 3-DHP-selenocysteine, di-gamma-glutamylselenocysteine, selenoglutaglutathione-gamma-glutamylcysteine, dehydroxy 5' -methylselenoadenosine, ethylselenoadenosine, seleno (hydroxy) -selenophenyl- (3' -deoxy-adenosine), allylselenadenosylhomocysteine, selenoadenosine homocysteine, selenoadenosine-Se (methyl) -selenoxide, adenosine-hydroxyselenoxide, ethylselenoadenosine, seleno- (hydroxy) -selenophenyl- (3' -deoxy-adenosine), adenosine-hydroxyselenoxide, and selenoadenosine-Se (methyl) -selenoxide.

In embodiments, any of the compounds described herein may be modified with prodrugs to extend half-life, protect the compound from oxidation, target the compound to a tissue, and/or allow the compound to pass through the blood-brain barrier.

In embodiments, the prodrug comprises a selenoglycoside. Glycosides include monosaccharides, disaccharides and oligosaccharides. The sugar may comprise ribose, glucose, galactose or mannose. For example, galactose conjugated to a selenium moiety may target the compound to the liver.

In other embodiments, the prodrug comprises selenazolidine. These compounds provide a slow release of the compound.

In still other embodiments, prodrugs comprise conjugation of a selenium organic compound described herein to a vitamin, e.g., C or E. These prodrug conjugates have improved protection.

In still other embodiments, the prodrug is a cytochrome P450 activated prodrug. For example, cyclic phosphates or phosphonates. In particular, nucleosides are modified with these molecules, and provide molecular targeting to the liver. Exemplary prodrugs include hepdi prodrug. Other embodiments of cytochrome P450 activated prodrugs improve bioavailability and are described in Huttunen et al Current Medicinal Chemistry 200815: 2346.

In embodiments, any of the compounds of formula (I), formula (II), formula (III) may be modified to reduce the oxidation of selenium. In embodiments, the compounds may form dimers through linkages between selenium atoms.

In embodiments, any of the compounds of formula (I), formula (II), formula (III) may be modified by the attachment of a tissue targeting agent or other agent for increasing the half-life of the compound. In embodiments, the tissue-targeting agent comprises an antibody that specifically binds to a tissue-specific antigen, transferrin receptor, or a prodrug as described herein.

In other embodiments, the compound may be linked to or combined with a polymeric carrier or nanoparticle carrier to deliver the composition to the brain and provide other tissue targeting. Such polymeric carriers include, but are not limited to, polyethylene glycol, polylactide, polyglycolide, polyorthoester, polyvinylpyrrolidone, and polyvinyl alcohol. Microspheres and liposomes include poly (lactic-co-glycolic acid) (PLGA) microspheres. Other nanoparticles include phospholipids, chitosan, lactic acid and dextran.

Lipid prodrugs may also be suitable for use with the compounds of the present invention. By way of non-limiting example, certain lipid prodrugs are described in Hostetler et al (1997 biochem. pharm. 53:1815-1822) and Hostetler et al 1996 Antiviral Research 31:59-67), both of which are incorporated herein by reference in their entirety. Other examples of suitable prodrug technologies are described in WO 90/00555, WO 96/39831, WO 03/095665a2, U.S. patent No. 5,411,947, 5,463,092, 6,312,662, 6,716,825, and U.S. patent application publication nos. 2003/0229225 and 2003/0225277, each of which is incorporated herein by reference in its entirety. Such prodrugs may also have the ability to target the pharmaceutical compound to a particular tissue within a patient, such as the liver, as described in Erion et al (2004 j. Am. chem. soc. 126:5154-5163; Erion et al Am. soc. pharm. & expert. ther. DOI:10.1124/jept.104.75903 (2004); WO 01/18013 a1; U.S. patent No. 6,752,981), each of which is incorporated herein by reference in its entirety. Other prodrugs of compounds suitable for use in the present invention are described, by way of non-limiting example, in WO 03/090690, U.S. Pat. No. 6,903,081, U.S. patent application No. 2005/0171060A1, U.S. patent application No. 2002/0004594A1, and Harris et al (2002 Antiviral Chem & Chemo. 12: 293-207300; Knaggs et al 2000 Bioorganic & Med. Chem. Letters 10: 2075-2078), each of which is incorporated herein by reference in its entirety.

In some embodiments, compositions are provided comprising one or more compounds each according to formula (I). In some aspects, a composition comprising one or more compounds each according to formula (I) comprises 5' -methylselenoadenosine, or a pharmaceutically acceptable salt, hydrate, or prodrug thereof; and 5' -selenadenosylhomocysteine or a pharmaceutically acceptable salt, hydrate or prodrug thereof.

In some embodiments, compositions are provided comprising one or more compounds according to each of formula (I) and formula (III). In some aspects, a composition comprising one or more compounds each according to formula (I) and formula (III) comprises 5' -methylselenoadenosine, or a pharmaceutically acceptable salt, hydrate, or prodrug thereof; 5' -selenadenosylhomocysteine or a pharmaceutically acceptable salt, hydrate or prodrug thereof; and gamma-glutamyl-methylseleno-cysteine or a pharmaceutically acceptable salt, hydrate or prodrug thereof.

In some embodiments, a composition comprising one or more compounds according to each of formula (I) and formula (III) comprises 5' -methylselenoadenosine, or a pharmaceutically acceptable salt, hydrate, or prodrug thereof; and gamma-glutamyl-methylseleno-cysteine or a pharmaceutically acceptable salt, hydrate or prodrug thereof.

In some embodiments, compositions are provided comprising one or more compounds according to each of formula (II) and formula (III). In some aspects, a composition comprising one or more compounds each according to formula (II) and formula (III) comprises 5' -selenadenosylhomocysteine or a pharmaceutically acceptable salt, hydrate or prodrug thereof; and gamma-glutamyl-methylseleno-cysteine or a pharmaceutically acceptable salt, hydrate or prodrug thereof.

According to another aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of one or more compounds of the present invention, or a pharmaceutically acceptable salt, ester or prodrug thereof, in association with a pharmaceutically acceptable diluent or carrier.

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) tragacanth powder; (5) malt; (6) gelatin; (7) talc powder; (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 glycerol, 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 hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) a ringer's solution; (19) ethanol; (20) a phosphate buffer solution; and (21) other non-toxic compatible materials used in pharmaceutical formulations.

The compositions may be formulated for any route of administration, in particular for oral, rectal, transdermal, subcutaneous, intravenous, intramuscular or intranasal administration. The compositions may be formulated in any conventional form, for example, as tablets, capsules, lozenges, solutions, suspensions, dispersions, syrups, sprays, gels, suppositories, patches and emulsions.

As is well known in the medical arts, the dosage to any one subject may depend on a number of factors, including the size of the patient, body surface area, age, the particular compound to be administered, sex, number and route of administration, general health, and interaction with other drugs being administered concurrently. The pharmaceutical compositions may be formulated and administered systemically or locally depending on the target sought to be altered by the treatment. Techniques for formulation and administration can be found in the latest version of "Remington's Pharmaceutical Sciences" (Mack Publishing Co, Easton Pa.). Suitable routes may include, for example, oral or transmucosal administration; and parenteral delivery, including intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, or intranasal administration.

Pharmaceutical compositions suitable for use herein include compositions in which the active ingredient (e.g., 5' -methylselenoadenosine, Se-adenosyl-L-homocysteine, gamma-glutamyl-methylselenocysteine, compounds of formula I, compounds of formula II, compounds of formula III, and combinations thereof) is included in an amount effective to achieve the intended purpose. For example, in a preferred embodiment, an effective amount of the pharmaceutical composition comprises an amount of a compound selected from the group consisting of 5' -methylselenoadenosine, Se-adenosyl-L-homocysteine, γ -glutamyl-methylselenocysteine, a compound of formula I, a compound of formula II, a compound of formula III, and combinations thereof. Determination of an effective amount is well within the ability of those skilled in the art, especially in light of the disclosure provided herein.

Selenized yeast containing 2% or less inorganic selenium typically contains many selenium-containing components. The main selenium components of a typical aqueous extract of selenized yeast are selenoprotein and selenomethionine. Other small molecular weight components having a molecular weight of less than 1000 kilodaltons ("Kda") have been identified, isolated and purified. Some of the components and compounds present in selenized yeast or aqueous extracts thereof, when isolated from such selenized yeast, have less desirable biological activity or even inhibitory biological activity on mitochondrial function. Compounds such as glutamyl selenocysteine and sulfur-containing compounds such as 5' -methylthioadenosine, S-adenosyl-L-homocysteine and gamma-glutamyl-methyl-cysteine are inactive or, in some cases, inhibitory, as described and shown herein.

Some selenium-containing compounds have been prepared synthetically, purified and screened in biological activity assays. The concentration range screened includes about 15 to about 500 ppb. Biological activity can be detected even at 15 ppb for the compositions described herein. In embodiments, the biological activity assay is a mitochondrial potential assay. When obtained from selenized yeast, not all of the components and compounds present in the yeast or aqueous extract thereof have biological activity, and some of the components and compounds inhibit the desired biological activity.

In embodiments, the synthetically produced selenium-containing compound is formulated in the composition at a different rate than is present in the aqueous extract. For example, a typical water extract has 7: 1 ratio of gamma-glutamyl-methylselenocysteine to 5' -methylselenoadenosine or Se-adenosyl-L-homocysteine. In embodiments, a composition containing a synthetically produced compound may comprise equal amounts of each selenium-containing compound, e.g., a ratio of at least 1:1:1 of γ -glutamyl-methylselenocysteine to 5' -methylselenocdenosine to Se-adenosyl-L-homocysteine. In other embodiments, the composition may comprise at least 2 components in a ratio of 5:1 to 1: 1.

Compositions comprising one or more compounds (including 5' -methylselenoadenosine, Se-adenosyl-L-homocysteine, gamma-glutamyl-methylseleno-cysteine, compounds of formula (I), compounds of formula (II), compounds of formula (III), and combinations thereof) can be administered intravenously to a subject (e.g., a patient) in a pharmaceutically acceptable carrier, such as physiological saline. Standard methods of intracellular delivery of the compounds (e.g., delivery via liposomes) can be used. Such methods are well known to those of ordinary skill in the art.

Compositions comprising selenium may be used for intravenous administration as well as parenteral administration, such as intravenous, subcutaneous, intramuscular, and intraperitoneal. For injection, the selenium-containing compositions (e.g., pharmaceutical compositions) of the present application may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks 'solution, ringer's solution, or physiological buffered saline. For tissue or cell administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

Compositions comprising 5' -methylselenoadenosine, Se-adenosyl-L-homocysteine, gamma-glutamyl-methylselenocysteine, compounds of formula (I), compounds of formula (II), compounds of formula (III), and combinations thereof, may be added to nutritional beverages or foods (e.g., ENSURE, POWERBAR, etc.), multivitamins, nutritional products, foods, and the like, for daily consumption.

In other embodiments, the compositions of the present application may be formulated in dosages suitable for oral administration using pharmaceutically acceptable carriers well known in the art. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral or nasal ingestion by a patient to be treated.

In some embodiments of the present application, a composition and/or formulation comprising selenium may be administered to a subject alone or in combination with other forms of selenium, drugs, small molecules, or in a pharmaceutical composition where it is mixed with excipients or other pharmaceutically acceptable carriers. In embodiments, the composition may include one or more amino acids or seleno-amino acids, such as methionine, cysteine, or selenocysteine, to minimize toxicity. In one embodiment of the present application, the pharmaceutically acceptable carrier is pharmaceutically inert. In another embodiment of the present application, a composition comprising selenium may be administered alone to an individual susceptible to, at risk of developing, or suffering from a disease or condition associated with mitochondrial dysfunction.

Methods of using compounds and compositions

As described herein, the compounds and combinations thereof can be used in a tissue-specific and tissue-appropriate manner to enhance mitochondrial function, treat mitochondrial dysfunction, treat alzheimer's disease, and modulate glucose metabolism.

A. Mitochondrial function and dysfunction

Mitochondria are the major energy-producing organelle in the cells of higher organisms. Mitochondria provide direct and indirect biochemical regulation of various cellular processes of respiration, oxidation, and metabolism. These include Electron Transport Chain (ETC) activity, which drives oxidative phosphorylation to produce metabolic energy in the form of Adenosine Triphosphate (ATP), and which is also the basis for important mitochondrial function in intracellular calcium homeostasis.

In addition to their role in the energy production of growing cells, mitochondria (or at least mitochondrial components) are involved in Programmed Cell Death (PCD), also known as apoptosis (see, e.g., Newmeyer et al Cell 1994, 79: 353-. Apoptosis is essential for the normal development of the nervous system and for the proper functioning of the immune system. In addition, some disease states are thought to be associated with insufficient or excessive levels of apoptosis (e.g., cancer and autoimmune diseases, and in the latter case stroke damage and neurodegeneration in alzheimer's disease, respectively). The role of mitochondria in apoptosis has been demonstrated (see, e.g., Green and Reed, Science, 1998, 281: 1309-.

Mitochondrial-related diseases (e.g., caused by mitochondrial dysfunction) may also be associated with loss of mitochondrial membrane electrochemical potential through mechanisms other than free radical oxidation, and permeability shifts may be due to direct or indirect effects of mitochondrial genes, gene products or related downstream mediator molecules and/or extramitochondrial genes, gene products or related downstream mediators, or other known or unknown causes. Thus, loss of mitochondrial potential may be an important event in the progression of diseases associated with altered mitochondrial function, including degenerative diseases as well as diseases/conditions associated with aging (e.g., cancer, cardiovascular disease and heart failure, type 2 diabetes, alzheimer's and parkinson's diseases, fatty liver disease, cataracts, osteoporosis, muscle atrophy, sleep disorders, and inflammatory diseases such as psoriasis, arthritis, and colitis). The methods as described herein can be used to enhance mitochondrial function, whether or not the cells in the subject are under stress or disease conditions.

The compounds and compositions disclosed herein exhibit tissue specificity with respect to their effect on mitochondrial function.

Embodiments include methods or uses for enhancing mitochondrial function in one or more renal cells, comprising: administering to a cell an effective amount of a composition comprising a compound selected from the group consisting of 5' -methylselenoadenosine, a compound of formula I, and mixtures thereof, wherein the effective amount enhances mitochondrial function as compared to a cell not treated with the composition. In further embodiments, one or more compounds may be isolated and/or purified.

In embodiments, the composition may exclude one or more of Se-adenosyl-L-homocysteine, gamma-glutamyl-methylselenocysteine, glutamyl selenocysteine, a compound of formula III, 5' -methylthioadenosine, or S-adenosyl-L-homocysteine.

In an embodiment, the present application provides the use of a composition comprising a compound selected from the group consisting of 5' -methylselenoadenosine, a compound of formula I, and mixtures thereof, wherein the effective amount enhances mitochondrial function in renal cells.

In embodiments, the composition comprises a compound selected from 5' -methylselenoadenosine and/or a compound of formula I. In embodiments, the composition for enhancing mitochondrial function in renal cells may not include one or more of Se-adenosyl-L-homocysteine, methylthioadenosine, or S-adenosyl-L-homocysteine.

Embodiments of the present application include methods or uses for enhancing mitochondrial function in one or more cells selected from skeletal muscle cells, neuronal cells, and combinations thereof, the method comprising: administering an effective amount of a composition comprising a compound selected from the group consisting of 5' -methylselenoadenosine, Se-adenosyl-L-homocysteine, γ -glutamyl-methylselenocysteine, a compound of formula I, a compound of formula II, a compound of formula III, and combinations thereof, to a cell, wherein the effective amount enhances mitochondrial function as compared to a cell not treated with the composition. In further embodiments, one or more compounds may be isolated and/or purified.

In an embodiment, the present application provides the use of a composition comprising a compound selected from the group consisting of 5' -methylselenoadenosine, Se-adenosyl-L-homocysteine, gamma-glutamyl-methylseleno-cysteine, a compound of formula I, a compound of formula II, a compound of formula III, and combinations thereof, wherein the effective amount enhances mitochondrial function in one or more cells selected from the group consisting of skeletal muscle cells, neuronal cells, and combinations thereof.

In embodiments, the composition comprises a compound selected from the group consisting of 5' -methylselenoadenosine, Se-adenosyl-L-homocysteine, a compound of formula I, a compound of formula II, and combinations thereof. In embodiments, the composition for enhancing mitochondrial function in muscle cells may not include one or more of methylthioadenosine or S-adenosyl-L-homocysteine. Although not intended to limit the scope of the present application, methylthioadenosine or S-adenosyl-L-homocysteine exhibit toxic effects on mitochondrial function in skeletal muscle cells.

The presence of certain sulfur-containing molecules in commonly used selenium supplements (e.g., selenium-enriched yeast) can, in some cases, inhibit mitochondrial activity and lead to pre-diabetic states over time. It is well documented in the literature that adult-onset diabetes is associated with a gradual decrease in mitochondrial activity over a period of years. This is particularly important in the case of skeletal muscle, which is estimated to use 75-80% of the daily glucose intake. Even a modest decrease in the ability of muscle mitochondria to effectively consume glucose (e.g., 20% inhibition) can lead to serious health problems over time. For example, a 2-fold stimulation of mitochondrial activity noted in skeletal muscle cells in response to the compound Se-adenosyl-L-homocysteine may represent a way to avoid or delay mitochondrial depletion in muscle tissue in pre-diabetic or diabetic subjects.

In embodiments, the composition for enhancing mitochondrial function in neuronal cells comprises a compound selected from the group consisting of 5' -methylselenoadenosine, Se-adenosyl-L-homocysteine, γ -glutamyl-methylselenocysteine, a compound of formula I, a compound of formula II, a compound of formula III, and combinations thereof. In further embodiments, one or more compounds may be isolated and/or purified. In embodiments, the composition for enhancing mitochondrial function in neuronal cells may not comprise one or more of selenomethionine or glutamyl selenocysteine.

In embodiments, the composition for enhancing mitochondrial function in neuronal cells comprises a compound selected from Se-adenosyl-L-homocysteine and/or a compound of formula II and at least one other selenium compound selected from 5' -methylselenoadenosine, γ -glutamyl-methylselenocysteine, a compound of formula I, a compound of formula III, and combinations thereof.

In embodiments, the composition for enhancing mitochondrial function in neuronal cells comprises a composition comprising a compound selected from the group consisting of 5' -methylthioadenosine, S-adenosyl-L-homocysteine, γ -glutamyl-methyl-cysteine, and combinations thereof. In embodiments, the neuronal cell targeting composition may comprise one or more sulfur analogs that increase mitochondrial activity in neuronal cells.

Other embodiments include methods or uses for enhancing mitochondrial function in one or more hepatocytes, comprising: administering an effective amount of a composition comprising at least three different compounds selected from the group consisting of 5' -methylselenoadenosine, Se-adenosyl-L-homocysteine, γ -glutamyl-methylselenocysteine, a compound of formula I, and a compound of formula III to a cell, wherein the effective amount enhances mitochondrial function compared to a cell not treated with the composition. In further embodiments, one or more compounds may be isolated and/or purified.

In an embodiment, the present application provides the use of a composition comprising at least three different compounds selected from the group consisting of 5' -methylselenoadenosine, Se-adenosyl-L-homocysteine, γ -glutamyl-methylselenocysteine, a compound of formula I, and a compound of formula III, for enhancing mitochondrial function in hepatocytes.

In embodiments, the composition for enhancing mitochondrial function in one or more hepatocytes comprises at least three compounds selected from the group consisting of 5' -methylselenoadenosine, Se-adenosyl-L-homocysteine, γ -glutamyl-methylselenocysteine, a compound of formula I, a compound of formula II, a compound of formula III, and combinations thereof, to enhance mitochondrial function in hepatocytes. In embodiments, the composition may exclude one or more compounds selected from the group consisting of 5' -methylthioadenosine, S-adenosyl-L-homocysteine, glutamyl-methyl-cysteine, and combinations thereof.

In embodiments, the increased mitochondrial function ranges from about a 50% increase to a 500% increase as compared to the same type of cell not treated with the compound. The magnitude of the response depends on the cell type, the particular compound, and the time of contact with the cell. In embodiments, the effect on mitochondrial function is about-66% to +200% compared to the same type of cell treated with one or more sulfur analogs of the selenium compound. In the case of some cell types, sulfur analogs have no measurable effect on mitochondrial activity, in the case of other cell types, they are inhibitory, and in the case of still other cell types, they are stimulatory.

In embodiments, the one or more compounds have a tissue-specific effect on the expression of the uncoupling protein. Uncoupling proteins (UCP) in Mitochondria (MT) are important for maintenance and thermogenesis of mitochondrial potential or integrity. Loss of UCP2 has been shown to result in decreased lifespan and increased production of Reactive Oxygen Species (ROS) in the MT (see, e.g., Am J Physiol Endocrinol Metab, Andrews et al, 2009.296 (4): p. E621-7; Curr Aging Sci, Andrews et al, 2010.3 (2): p. 102-12). However, because UCPs decouple electron transport in mitochondria, they have the net effect of reducing ATP production from glucose in the cell. It is well known that mitochondrial ATP production is critical for glucose-stimulated insulin secretion (GSIS) by pancreatic β -cells. Indeed, it has been shown that inhibition of one UCP, specifically UCP2, reverses diet-induced diabetes by positively affecting both insulin secretion and action (De Souza et al FASEB J, 2007, 21(4): 1153-.

In embodiments, a method or use of down-regulating Ucp2 and/or Ucp3 gene expression in a neuronal cell comprises: administering an effective amount of a composition comprising a compound selected from the group consisting of 5' -methylselenoadenosine, Se-adenosyl-L-homocysteine, γ -glutamyl-methylselenocysteine, a compound of formula I, a compound of formula II, a compound of formula III, and combinations thereof, to a cell, wherein the effective amount down-regulates expression of Ucp2 and/or Ucp3 in neuronal cells compared to a cell not treated with the composition. In further embodiments, one or more compounds may be isolated and/or purified.

In an embodiment, the present application provides for the use of a composition comprising a compound selected from the group consisting of 5' -methylselenoadenosine, Se-adenosyl-L-homocysteine, γ -glutamyl-methylseleno-cysteine, a compound of formula I, a compound of formula II, a compound of formula III, and combinations thereof, for down-regulating Ucp2 and/or Ucp3 gene expression in a neuronal cell.

In other embodiments, a method or use of down-regulating Ucp2 gene expression in a hepatocyte comprises: administering to a cell an effective amount of a composition comprising at least three compounds selected from the group consisting of 5' -methylselenoadenosine, Se-adenosyl-L-homocysteine, γ -glutamyl-methylseleno-cysteine, a compound of formula I, a compound of formula II, a compound of formula III, and combinations thereof, wherein the effective amount down-regulates Ucp2 expression in hepatocytes compared to cells not treated with the composition. In further embodiments, one or more compounds may be isolated and/or purified.

In an embodiment, the present application provides the use of a composition comprising at least three different compounds selected from the group consisting of 5' -methylselenoadenosine, Se-adenosyl-L-homocysteine, γ -glutamyl-methylseleno-cysteine, a compound of formula I, and a compound of formula III, for down-regulating Ucp2 and/or Ucp3 gene expression in a hepatocyte.

Methods for determining gene expression in a cell are known to those skilled in the art and include hybridization to probes (e.g., on an array), or by PCR methods. Arrays and/or primers for determining gene expression are commercially available. Using the exemplary sequences for Ucp1, Ucp2, and/or Ucp3, primers can be readily designed. Ucp1 are found in NM _021833/gI194733736, Ucp2 is found in NM _003355/gI13259540 and Ucp3 is found in NM _003356/gI 215273349.

In embodiments, an effective amount of a compound and composition as described herein is an amount effective to enhance mitochondrial function without being toxic to cells. Enhanced mitochondrial function can be determined using a variety of assays on samples obtained from subjects treated according to the compositions described herein. The effective amount is selected to be non-toxic to any of the exemplified cells including kidney cells, mouse skeletal cells, human neuronal cells, or mouse liver cells.

In embodiments, an effective amount of a composition administered to a subject to enhance mitochondrial function in one or more kidney, neuronal, liver or skeletal muscle cells is about at least 5ug or more, or 800 ug or less of a single desired bioactive selenium-containing compound or a plurality of desired bioactive selenium-containing compounds per day. When a plurality of desired bioactive selenium-containing compounds are present, the effective amount of the composition is the total of all desired bioactive selenium-containing compounds in the composition.

In embodiments, an effective amount administered to a subject is about 5ug to about 800 ug and each numerical value therein, 5ug to about 700 ug and each numerical value therein, 5ug to about 600 ug and each numerical value therein, 5ug to about 500 ug and each numerical value therein, 5ug to about 400 ug and each numerical value therein, 5ug to about 300 ug and each numerical value therein, 5ug to about 200 ug and each numerical value therein, 5ug to about 100 ug and each numerical value therein, or 5ug to 50 ug and each numerical value therein.

In embodiments of the composition comprising a biologically active selenium-containing compound in need thereof as described herein, the effective amount administered to the subject is preferably 200 ug or less per day or 50 ug or less per day. In embodiments, the dosage may be adjusted according to efficacy or whether any overt signs of selenium poisoning, such as garlic-flavoured respiration, hair loss or cuticle, are observed in the subject.

In embodiments, in a composition comprising at least two or more compounds selected from the group consisting of 5' -methylselenoadenosine, Se-adenosyl-L-homocysteine, gamma-glutamyl-methylseleno-cysteine, a compound of formula (I), a compound of formula (II), a compound of formula (III), and combinations thereof, the compounds are present in equal proportions in the composition. In other embodiments, the ratio of one desired bioactive selenium-containing compound to another may be about 4:1 to 1: 1.

In embodiments, the dose is administered at least once daily for a period of time to achieve a steady state of elemental selenium in the blood. In embodiments, the dose is administered daily for at least 60 or 90 days. In embodiments, the dose is administered while the subject is experiencing symptoms of the disease or disorder. In embodiments, the subject is at an age at which the risk of a mitochondrial-related disease is increased, e.g., at least 40 years of age.

The methods of the present application can be used to treat (e.g., prophylactically or therapeutically) a subject (e.g., a subject having a condition associated with mitochondrial dysfunction). While not intending to limit the scope of the present disclosure, evidence has been provided demonstrating that the three synthetic organoselenium compounds, alone or in various combinations, have the ability to significantly increase mitochondrial activity in different cell types, namely, kidney cells, skeletal muscle cells, neuronal cells, and liver cells. Mechanistically, regulation of UCP may provide an explanation for this increase, and we provide evidence below that expression of other proteins critical to mitochondrial function and biogenesis may also be favorably affected by these compounds. However, regardless of the mechanism of action, the fact that these compounds can stimulate mitochondrial activity in a trans-tissue manner means that they may be particularly valuable in ameliorating the development and progression of seemingly diverse diseases such as Alzheimer's Disease (AD) and type 2 diabetes (T2 DM).

A composition comprising one or more compounds (including 5' -methylselenoadenosine, Se-adenosyl-L-homocysteine, gamma-glutamyl-methylselenocysteine, a compound of formula I, a compound of formula II, a compound of formula III, and combinations thereof) can be administered to a subject (e.g., a patient) in any of the manners described herein.

In embodiments delivered to skeletal muscle, the compositions as described herein may be topically applied in the form of a gel, patch, or cream.

In other embodiments, delivery to the brain may be targeted by using liposomes or PLGA spheres with antibodies, transferrin receptors, or prodrugs as targeting agents.

In other embodiments, delivery to the liver can be targeted using targeted prodrugs as described herein, including hepdi ect or selenoglycoside.

In another embodiment of the present application, a composition comprising a selenium organic compound described herein can be administered alone to an individual who is susceptible to, at risk of developing, or suffering from a disease or condition associated with mitochondrial dysfunction. Such diseases include diabetic nephropathy, Alzheimer's disease and type II diabetes.

A. Alzheimer's disease

Alzheimer's disease ("AD") is the 6 th leading cause of death in the united states ("USA") and is the most common form of dementia. The disease is currently estimated to affect 510 million years of age in the USA in people 65 years and older. AD is characterized histopathologically by two marker lesion A β plaques (see Kumar et al 2000) and by NFT consisting of a highly-phosphorylated form of the microtubule-associated protein tau (see Dunckley et al, published on-line 2005, 10.2005, 19.2005 and paragraph 155-163 of published patent application 201110038889 AI). There is a lot of evidence in the literature that both a β and NFT are important partners in the pathogenesis of alzheimer's disease, and they each and consistently act to maximize cognitive impairment and neuronal loss in affected individuals. Mutations in Amyloid Precursor Protein (APP) result in AD with 100% penetration, and familial AD (fad) -related mutations in APP, presenilin-i (PSEN), and presenilin-2 (PSEN-2) result in increased levels of amyloid β production and a β aggregation.

In addition, APP-overexpressed mice (APP-Tg) showed Α β deposition and memory impairment, did not form NFTs or suffered neuronal loss. Plaques are formed when Amyloid Precursor Protein (APP) is abnormally processed by two enzymes, termed beta-secretase and y-secretase, resulting in the formation of the 39-42 amino acid peptide of beta amyloid. Gamma-secretase is in fact a multienzyme complex that includes presenilin-i (PSEN) and presenilin-2 (PSEN-2) as two key components.

A β aggregation is associated with memory loss in the AD model, and decreased APP expression reverses this loss (see Kumar et al Peptides 21: 17692000). Thus, reduced APP expression (at the gene or protein level) is understood by the skilled person as a method to combat age-dependent or neurodegenerative disorders involving memory loss, such as AD.

Modulation or inhibition of PSEN, PSEN-2, Nicastrin (which controls protein trafficking in the γ -secretase complex) is an important goal of AD therapy research, and some significant success has been published. For example, highly specific inhibitors that modulate PSEN activity in human neurons not only affect a β production, but also the Notch signal transduction pathway (see Seiffert et al J biol. chem. 275: 340862000). This is accompanied by changes in neurite morphology and suggests that modulation of gamma secretase PSEN-1 activity has a clinically beneficial effect on the neurite pathology of AD (see fig. Figueroa et al Neurobiology dis. 9: 492002). Furthermore, down-regulation of PSEN suggests a reduction in a β protein secretion (see Luo et al Acta pharmacol. sin.25: 16132004), and inhibition of PSEN was found to significantly improve memory in SAMP8 mice (see Kumar et al j.exp. biol. 212: 4942009).

Thus, a β is toxic to neurons, promotes neuronal cell death, and a β levels in the brain can be altered by modulating the level/activity of its precursor APP or the enzyme complex that processes APP. The skilled artisan will readily understand and appreciate that agents capable of inhibiting APP expression and/or APP addition to a β (e.g., B or γ secretases) are therapeutic targets for AD therapy.

The selenium-containing compounds and compositions disclosed herein affect gene expression of genes involved in B amyloid processing and tau phosphorylation. In addition, such compounds and compositions exhibit tissue specificity with respect to gene expression of genes involved in transcriptional activators.

In embodiments, a method or use for inhibiting B amyloid accumulation in one or more neuronal cells comprises: administering an effective amount of a composition comprising a compound selected from the group consisting of 5' -methylselenoadenosine, a compound of formula (I), and mixtures thereof, to one or more neuronal cells, wherein the effective amount inhibits B amyloid accumulation in neuronal cells compared to neuronal cells not treated with the composition.

In embodiments, a method or use for inhibiting B amyloid accumulation in one or more neuronal cells comprises: administering an effective amount of a composition comprising at least three different compounds selected from the group consisting of 5' -methylselenoadenosine, Se-adenosyl-L-homocysteine, γ -glutamyl-methylseleno cysteine, a compound of formula (I), and a compound of formula (III) to one or more neuronal cells, wherein the effective amount inhibits B amyloid accumulation in neuronal cells compared to neuronal cells not treated with the composition. In further embodiments, one or more compounds may be isolated and/or purified.

In embodiments, the present application provides the use of a composition comprising a compound selected from the group consisting of 5 ' -methylselenoadenosine, a compound of formula (I), or at least three different compounds selected from the group consisting of 5 ' -methylselenoadenosine, Se-adenosyl-L-homocysteine, γ -glutamyl-methylseleno-cysteine, a compound of formula (I), and a compound of formula (III), 5 ' -methylselenoadenosine, a compound of formula I, and mixtures thereof, for inhibiting B amyloid accumulation in neuronal cells.

In an embodiment, the composition comprises a compound selected from the group consisting of 5' -methylselenoadenosine, a compound of formula (I), and mixtures thereof, to inhibit the expression of presenilin 1 (PSEN) and Nicastrin in neuronal cells. In embodiments, the neuronal cell is an IMR 32 cell. IMR 32 cells are human neuronal cells that are models of alzheimer's disease. (Neill et al J. Neuroscience Res. 199439: 482). In embodiments, the composition for inhibiting gene expression in a neuronal cell may not include one or more of Se-adenosine-L-homocysteine or methylthioadenosine.

In embodiments, the present application provides the use of a composition comprising at least three different compounds selected from the group consisting of 5 ' -methylselenoadenosine, a compound of formula (I), or selected from the group consisting of 5 ' -methylselenoadenosine, Se-adenosyl-L-homocysteine, γ -glutamyl-methylseleno cysteine, a compound of formula (I), and a compound of formula (III), 5 ' -methylselenoadenosine, a compound of formula I, and mixtures thereof, for inhibiting the expression of presenilin 1 and nicastrin in neuronal cells.

In an embodiment, the composition comprises a compound selected from the group consisting of 5' -methylselenoadenosine, Se-adenosyl-L-homocysteine, γ -glutamyl-methylselenocysteine, a compound of formula (I), a compound of formula (II), a compound of formula (III), and combinations thereof, to inhibit gene expression of nicastrin and presenilin 1 in neuronal cells. In embodiments, the composition for inhibiting gene expression in a neuronal cell may not comprise one or more of selenomethionine or glutamyl selenocysteine.

In embodiments, the composition comprises 5' -methylselenoadenosine and/or a compound of formula (I) and at least one other selenium compound selected from Se-adenosyl-L-homocysteine, gamma-glutamyl-methylselenocysteine, a compound of formula (II), a compound of formula (III), and combinations thereof. In further embodiments, one or more compounds may be isolated and/or purified.

In embodiments, the composition comprises at least three compounds selected from the group consisting of 5' -methylselenoadenosine, Se-adenosyl-L-homocysteine, gamma-glutamyl-methylselenocysteine, a compound of formula (I), a compound of formula (II), a compound of formula (III), and combinations thereof, to inhibit gene expression in neuronal cells.

In embodiments, a method or use for inhibiting tau phosphorylation in one or more neuronal cells comprises: administering an effective amount of a composition comprising a compound selected from the group consisting of 5' -methylselenoadenosine, a compound of formula (I), and mixtures thereof, to one or more neuronal cells, wherein the effective amount inhibits tau phosphorylation in neuronal cells compared to neuronal cells not treated with the composition.

In embodiments, a method or use for inhibiting tau phosphorylation in one or more neuronal cells comprises: administering an effective amount of a composition comprising at least three different compounds selected from the group consisting of 5' -methylselenoadenosine, Se-adenosyl-L-homocysteine, gamma-glutamyl-methylselenocysteine, a compound of formula (I), and a compound of formula (III) to one or more neuronal cells, wherein the effective amount inhibits tau phosphorylation in the neuronal cells compared to neuronal cells not treated with the composition. In further embodiments, one or more compounds may be isolated and/or purified.

In embodiments, the composition comprises a compound selected from 5' -methylselenoadenosine and/or a compound of formula I to inhibit expression of Gsk3B in neuronal cells. In embodiments, the composition for inhibiting the expression of the Gsk3B gene in neuronal cells may not include one or more of Se-adenosyl-L-homocysteine or methylthioadenosine.

In embodiments, the composition comprises a compound selected from the group consisting of 5' -methylselenoadenosine, Se-adenosyl-L-homocysteine, gamma-glutamyl-methylselenocysteine, a compound of formula I, a compound of formula II, a compound of formula III, and combinations thereof to inhibit tau phosphorylation in neuronal cells. In embodiments, the composition for inhibiting tau phosphorylation in a neuronal cell may not include one or more of methionine or other sulfur-containing compounds.

In embodiments, the composition comprises at least two compounds selected from the group consisting of 5' -methylselenoadenosine, γ -glutamyl-methylselenocysteine, a compound of formula I, a compound of formula III, and combinations thereof to inhibit tau phosphorylation in neuronal cells.

In embodiments, the composition comprises a compound selected from gamma-glutamyl-methylselenocysteine and/or a compound of formula (III) to inhibit total tau in neuronal cells. In embodiments, a composition for inhibiting total tau in a neuronal cell may not include one or more of Se-adenosyl-L-homocysteine or methylthioadenosine.

In embodiments, a method or use for inhibiting FOXO phosphorylation in one or more neuronal cells comprises: administering an effective amount of a composition comprising a compound selected from the group consisting of 5' -methylselenoadenosine, Se-adenosyl-L-homocysteine, gamma-glutamyl-methylselenocysteine, a compound of formula (I), a compound of formula (III), and combinations thereof, to one or more neuronal cells, wherein the effective amount inhibits FOXO phosphorylation compared to cells not treated with the composition. In further embodiments, one or more compounds may be isolated and/or purified.

In an embodiment, the present application provides the use of a composition comprising a compound selected from the group consisting of 5' -methylselenoadenosine, Se-adenosyl-L-homocysteine, γ -glutamyl-methylseleno-cysteine, a compound of formula (I), a compound of formula (III), and combinations thereof, for inhibiting FOXO phosphorylation in neuronal cells.

In embodiments, a method or use for inhibiting FOXO phosphorylation in one or more neuronal cells while increasing FOXO phosphorylation in one or more hepatic cells comprises: administering to the liver and neuronal cells an effective amount of a composition comprising at least three compounds selected from the group consisting of 5' -methylselenoadenosine, Se-adenosyl-L-homocysteine, gamma-glutamyl-methylseleno-cysteine, a compound of formula (I), a compound of formula (III), and combinations thereof, wherein the effective amount reduces FOXO phosphorylation in neuronal cells and increases FOXO phosphorylation in liver cells as compared to cells not treated with the composition.

In embodiments, the composition comprises a compound selected from the group consisting of 5' -methylselenoadenosine, Se-adenosyl-L-homocysteine, γ -glutamyl-methylseleno-cysteine, a compound of formula (I), a compound of formula (II), a compound of formula (III), and combinations thereof to inhibit phosphorylation of FOXO3 and/or FOXO4 in a neuronal cell. In embodiments, a composition for inhibiting FOXO3 and/or FOXO4 phosphorylation in a neuronal cell may not comprise methionine or selenomethionine. In embodiments, the composition comprises 5' -methylselenoadenosine and/or a compound of formula (I) and at least one other selenium compound selected from Se-adenosyl-L-homocysteine, gamma-glutamyl-methylselenocysteine, a compound of formula (II), a compound of formula (III), and combinations thereof.

In an embodiment, the composition comprises at least three compounds selected from the group consisting of 5' -methylselenoadenosine, Se-adenosyl-L-homocysteine, γ -glutamyl-methylselenocysteine, a compound of formula (I), a compound of formula (II), a compound of formula (III), and combinations thereof to inhibit phosphorylation of FOXO 3 and FOXO 4 in neuronal cells.

In an embodiment, the present application provides the use of a composition comprising a compound selected from the group consisting of 5' -methylselenoadenosine, Se-adenosyl-L-homocysteine, γ -glutamyl-methylseleno-cysteine, a compound of formula (I), a compound of formula (II), a compound of formula (III), and combinations thereof, to increase expression of PGC1a in neuronal cells.

In embodiments, a method or use of increasing expression of PGC1a in one or more neuronal cells comprises: administering an effective amount of a composition comprising at least three different compounds selected from the group consisting of 5' -methylselenoadenosine, Se-adenosyl-L-homocysteine, γ -glutamyl-methylseleno-cysteine, a compound of formula (I), and a compound of formula (III) to one or more neuronal cells, wherein the effective amount increases PGC1a expression compared to cells not treated with the composition.

In embodiments, the method or use for increasing expression of PGC1a in one or more neuronal cells and not affecting expression of PGC1a in one or more hepatocytes comprises: administering to the liver and neuronal cells an effective amount of a composition comprising at least three compounds selected from the group consisting of 5' -methylselenoadenosine, Se-adenosyl-L-homocysteine, gamma-glutamyl-methylseleno-cysteine, a compound of formula (I), a compound of formula (III), and combinations thereof, wherein the effective amount increases expression of PGC1a in the one or more neuronal cells and does not affect expression of PGC1a in the one or more liver cells, as compared to cells not treated with the composition.

In embodiments, the composition comprises a compound selected from 5' -methylselenoadenosine and/or a compound of formula I and at least one other selenium compound selected from Se-adenosyl-L-homocysteine, gamma-glutamyl-methylselenocysteine, a compound of formula II, a compound of formula III, and combinations thereof.

In embodiments, the composition comprises at least three compounds selected from the group consisting of 5' -methylselenoadenosine, Se-adenosyl-L-homocysteine, γ -glutamyl-methylselenocysteine, a compound of formula (I), a compound of formula (II), a compound of formula (III), and combinations thereof to increase expression of PGC1a in neuronal cells.

In embodiments, PGC1a expression is increased, FOXO phosphorylation is decreased, PSEN1 is decreased, nicastrin is decreased, Gsk3b is decreased in a range of about 50% increase or decrease to 500% increase or decrease, respectively, as compared to the same type of cell not treated with the compound. The magnitude of the response depends on the cell type, the particular compound, and the time of contact with the cell.

The effect of the compounds and compositions of the present application on the phosphorylation of FOXO3 and FOXO4 and on the expression of PGC1a shows tissue specificity. Although not intended to limit the present application, inhibition of FOXO3 and FOXO4 phosphorylation in neuronal cells allows FOXO to enter the nucleus and activate transcription. PGC1a is also a transcriptional activator that regulates energy metabolism (including gluconeogenesis) in cells. FOXO and PGC1a promote transcriptional activation of genes such as glucose-6-phosphatase (G6 pc). The increase in gluconeogenesis confers to neuronal cells the ability to produce glucose and maintain energy stores.

As described above, the production of a β and phosphorylated tau significantly contributes to the pathology of alzheimer's disease. The results provided herein provide evidence that the compounds and compositions described herein affect the gene expression of presenilin 1 and nicastrin. Both enzymes have been shown to be involved in the production of a β. Furthermore, inhibition of phosphorylation of tau and/or total tau demonstrates that compounds and compositions as described herein also affect the production of NFT. Furthermore, the effect of the compounds on enhancing mitochondrial function, increasing FOXO activation and increasing PGC1a gene expression provides neuronal cells with increased mitochondrial function, which can also be used for the treatment of alzheimer's disease by minimizing oxidative stress. Previous studies have shown that App transgenic mice fed selenium enriched yeast rather than the normal diet or the selenium deficient diet reduce the production of B amyloid plaques (Lovell et al Free rad. biol. med. 46: 15272009).

Methods for determining gene expression in a cell are known to those skilled in the art and include hybridization to probes (e.g., on an array) or by PCR methods. Arrays and/or primers for detecting gene expression are commercially available. Primers can be readily designed using the exemplary sequences for PSEN 1, PSEN2, Nicastrin, and Gsk3 b. Exemplary sequences of PSEN 1 are found at NM-000021/NM-007318, PSEN2 at NM-000447/NM-012486, Nicasctrin at NM-015331, and Ucp3 at NM-002093.

In embodiments, a method or use for treating alzheimer's disease comprises: administering to the subject an effective amount of a composition comprising a compound selected from the group consisting of 5' -methylselenoadenosine, a compound of formula (I), and combinations thereof. In embodiments, the composition for treating alzheimer' S disease may not include one or more of methylthioadenosine or S-adenosyl-L-homocysteine.

In embodiments, a method or use for treating alzheimer's disease comprises: administering a compound selected from the group consisting of 5' -methylselenoadenosine, Se-adenosyl-L-homocysteine, gamma-glutamyl-methylselenocysteine, a compound of formula (I), a compound of formula II, a compound of formula III, and combinations thereof. In embodiments, the composition for treating alzheimer's disease may not include methionine or selenomethionine.

In embodiments, the present application provides the use of a composition comprising a compound selected from the group consisting of 5 '-methylselenoadenosine, Se-adenosyl-L-homocysteine, gamma-glutamyl-methylselenocysteine, a compound of formula (I), a compound of formula (II), a compound of formula (III), and combinations thereof, to treat alzheimer's disease.

In embodiments, the method or use of treating alzheimer's disease further comprises selecting a composition that treats the symptoms of alzheimer's disease without producing adverse effects on glucose metabolism in the liver. In embodiments, the composition comprises 5' -methylselenoadenosine and/or a compound of formula (I). In embodiments, the composition may exclude one or more of Se-adenosyl-L-homocysteine, gamma-glutamyl-methylselenocysteine, a compound of formula (II), or a compound of formula (III). In embodiments, the composition comprises at least three compounds selected from the group consisting of 5' -methylselenoadenosine, Se-adenosyl-L-homocysteine, γ -glutamyl-methylselenocysteine, a compound of formula (I), a compound of formula (II), a compound of formula (III), and combinations thereof. In embodiments, the composition excludes one or more of H (5' -methylthioadenosine), I (S-adenosyl-L-homocysteine), or J (γ -glutamyl-methyl-cysteine).

In an embodiment, the composition comprises at least three compounds selected from the group consisting of 5 '-methylselenoadenosine, Se-adenosyl-L-homocysteine, gamma-glutamyl-methylseleno-cysteine, a compound of formula (I), a compound of formula (II), a compound of formula (III), and combinations thereof, for use in treating alzheimer's disease.

In embodiments, an effective amount of a compound and composition as described herein is an amount effective to treat alzheimer's disease without toxicity to the cells. The effective amount is selected to be non-toxic to any of the exemplified cells including kidney cells, mouse skeletal cells, human neuronal cells, or mouse liver cells.

In embodiments, an effective amount of a compound and composition as described herein is an amount effective to ameliorate symptoms of alzheimer's disease and/or modulate gene expression as described herein without toxicity to the cell. Modulation of gene expression in neuronal cells can be determined using a variety of assays as described herein on samples obtained from subjects treated according to the compositions described herein.

In embodiments, an effective amount of a composition for treating alzheimer's disease, inhibiting B amyloid processing, and/or tau phosphorylation in neuronal cells is about 5ug or more or 800 ug or less of a single desired bioactive selenium-containing compound or a plurality of desired bioactive selenium-containing compounds per day. When a plurality of desired bioactive selenium-containing compounds are present, an effective amount of the composition is the total of all compounds in the composition administered to the subject.

In embodiments, an effective amount administered to a subject within one day is about 5ug to about 800 ug and each numerical value therein, 5ug to about 700 ug and each numerical value therein, 5ug to about 600 ug and each numerical value therein, 5ug to about 500 ug and each numerical value therein, 5ug to about 400 ug and each numerical value therein, 5ug to about 300 ug and each numerical value therein, 5ug to about 200 ug and each numerical value therein, 5ug to about 100 ug and each numerical value therein, and 5ug to 50 ug and each numerical value therein.

In embodiments, the effective amount of a composition comprising a biologically active selenium-containing compound in need thereof as described herein administered to a subject is preferably 200 ug or less per day or 50 ug or less per day. In embodiments, the dosage may be adjusted according to efficacy or whether any significant signs of selenium poisoning, such as garlic-flavoured respiration, hair loss or cuticle, are observed in the subject.

In embodiments, in a composition comprising two or more compounds selected from the group consisting of 5' -methylselenoadenosine, Se-adenosyl-L-homocysteine, γ -glutamyl-methylselenocysteine, a compound of formula (I), a compound of formula (II), a compound of formula (III), and combinations thereof, the compounds are present in the composition in the same ratio. In other embodiments, the ratio of one compound to another may be about 4:1 to 1: 1.

In embodiments, the dose is administered at least once a day for a period of time to achieve a steady state of elemental selenium in the blood. In embodiments, the dose is administered daily for at least 60 or 90 days. In embodiments, the dose is administered while the subject is experiencing symptoms of the disease or disorder. In embodiments, the subject is at an age where the risk of a mitochondrial-related disease is increased, e.g., at least 40 years of age.

The methods of the present application can be used to treat (e.g., prophylactically or therapeutically) a subject (e.g., a subject having a condition associated with alzheimer's disease, B amyloid processing, tau phosphorylation and presenilin 1 gene expression, nicastrin, PGC1a and tau and FOXO3 and FOXO 4 phosphorylation). Compositions comprising one or more compounds (including 5' -methylselenoadenosine, Se-adenosyl-L-homocysteine, gamma-glutamyl-methylseleno-cysteine, compounds of formula (I), compounds of formula (II), compounds of formula (III), and combinations thereof) can be administered intravenously to a subject (e.g., a patient) in a pharmaceutically acceptable carrier, such as physiological saline. Standard methods for intracellular delivery of the compounds (e.g., delivery via liposomes) can be used. Such methods are well known to those of ordinary skill in the art. Compositions comprising selenium may be used for intravenous administration as well as parenteral administration, such as intravenous, subcutaneous, intramuscular, and intraperitoneal.

In embodiments, the compositions of the invention are formulated to cross the blood brain barrier. The compositions of the present invention may be combined with implant materials suitable for delivery to the brain, such as polymeric biodegradable implants, for example ethylene vinyl acetate. Other types of targeting may include receptor-mediated trafficking, such as insulin receptor and transferrin receptor. These receptors may be incorporated into liposomes or microspheres that also include compositions as described herein. In other embodiments, delivery to the brain may be targeted by using liposomes or PLGA spheres with antibodies, transferrin receptors, or prodrugs as targeting agents.

Thus, in some embodiments of the present application, a composition and/or formulation comprising selenium may be administered to a subject alone or in combination with other forms of selenium, drugs, small molecules, or in a pharmaceutical composition where it is mixed with an excipient or other pharmaceutically acceptable carrier. In one embodiment of the present application, the pharmaceutically acceptable carrier is pharmaceutically inert. In another embodiment of the present application, a composition comprising selenium may be administered alone to an individual susceptible to, at risk of developing, or suffering from a disease or condition associated with B amyloid processing or tau phosphorylation. Compositions comprising 5' -methylselenoadenosine, Se-adenosyl-L-homocysteine, gamma-glutamyl-methylseleno-cysteine, compounds of formula (I), compounds of formula (II), compounds of formula (III), and combinations thereof, can be added to nutritional beverages or foods (e.g., ENSURE, POWERBAR, etc.), multivitamins, nutritional products, foods, and the like, for daily consumption.

Glucose metabolism

Non-insulin dependent (type II) Diabetes Mellitus (DM) is a disease characterized by insulin resistance in skeletal muscle, liver and fat, and a deficiency in insulin secretion due to pancreatic β -cell function. Insulin resistance is an important feature of type II diabetes. For example, the vast majority of type II diabetes is known to be insulin resistant. Also, insulin resistance in the offspring of type II diabetes is the best predictor of later development of the disease (see, e.g., Warram et al Ann Intern Med 113: 9091990). Interventions that reduce insulin resistance also prevent diabetes from occurring. Optimal mitochondrial function is required for the secretion of normal glucose-stimulated insulin from pancreatic beta cells.

Skeletal muscle and liver are two important insulin-responsive organs in maintaining glucose homeostasis. The conversion of these organs to the insulin-resistant state accounts for most of the glucose metabolism changes seen in type II diabetic patients (see, e.g., Lowell and Shulman, Science 21: 3072005). In both organs, skeletal muscle is more important in terms of the consequences derived from the development of insulin resistance. This is because skeletal muscle has been found to process or metabolize 80-90% of the daily glucose intake (see, e.g., Freonzo et al 1985).

It has been demonstrated by genome-wide expression analysis that mitochondrial oxidative phosphorylation (OXPHOS) genes show reduced expression in prediabetic and diabetic individuals compared to healthy controls, and in many cases these genes are targets for the transcriptional co-activator, the proliferator-activated receptor gamma co-activator 1-alpha (PGC 1-alpha, see, e.g., mooth et al 2003). In these studies, the typical decrease in OXPHOS gene expression was modest (approximately 20%), but very consistent, with 89% of the genes studied showing lower expression in individuals with impaired glucose tolerance or type II diabetes relative to individuals with normal glucose tolerance.

It is generally understood and appreciated in the art that drugs or agents that increase OXPHOS activity in muscle exist as valuable therapies for type 2 diabetes. In support of this hypothesis, hyperkinetic was long known to be the best non-pharmacological intervention for the treatment of diabetes, as it increases mitochondrial activity and number and promotes OXPHOS gene expression.

In the liver, FOXO, in an activated or unphosphorylated state, is located in the nucleus where it binds to the promoter region of glucose 6-phosphatase and other factors such as PGC-1a, increasing transcription of glucose-6-phosphatase, thereby increasing the rate of glucose production. Glucose 6-phosphatase catalyzes the final step of gluconeogenesis and glycogenolysis, resulting in the release of glucose from the liver. Therefore, it is important in controlling glucose homeostasis, particularly in diabetic subjects.

Normally, the processing of FOXO phosphorylation is directly controlled by another kinase called AKT (protein kinase B). AKT phosphorylates FOXO and drives it away from the nucleus, thereby reducing glucose production by reducing the transcription rate of glucose 6-phosphatase. AKT itself is under downstream control through a small molecular cascade that begins with the binding of insulin to its receptor on the cell surface. This initiates a series of events including two other kinases, phosphatidylinositol 3-kinase (PI3K) and phosphoinositide-dependent protein kinase 1 (PDK 1). The entire pathway, termed the insulin/PI 3K/PDK1/Akt pathway, has the effect of controlling glucose homeostasis through insulin signaling. Under FOXO control, PDK1 phosphorylates and activates Akt, which in turn phosphorylates and inactivates FOXO.

In an embodiment, there is provided a method or use for increasing FOXO 3 and FOXO 4 phosphorylation in one or more hepatocytes comprising administering a composition comprising at least three compounds selected from the group consisting of 5' -methylselenoadenosine, Se-adenosyl-L-homocysteine, γ -glutamyl-methylselenocysteine, a compound of formula I, a compound of formula II, a compound of formula III, and combinations thereof.

In embodiments, a method of modulating glucose metabolism in one or more cells selected from the group consisting of hepatocytes, skeletal muscle cells, and combinations thereof comprises: administering to the one or more cells an effective amount of a composition comprising at least three different compounds selected from the group consisting of 5' -methylselenoadenosine, Se-adenosyl-L-homocysteine, gamma-glutamyl-methylseleno-cysteine, a compound of formula (I), and a compound of formula (III), wherein the effective amount alters glucose metabolism in hepatocytes or muscle cells as compared to cells not treated with the composition. In embodiments, the composition may exclude one or more of H (5' -methylthioadenosine), I (S-adenosyl-L-homocysteine), and/or J (γ -glutamyl-methyl-cysteine).

In an embodiment of the present application, there is provided the use of a composition comprising at least three different compounds selected from the group consisting of 5' -methylselenoadenosine, Se-adenosyl-L-homocysteine, γ -glutamyl-methylseleno-cysteine, a compound of formula (I) and a compound of formula (III) for modulating glucose metabolism in hepatocytes or muscle cells.

In embodiments, the method of treating type II diabetes comprises: administering to a subject an effective amount of a composition comprising at least three different compounds selected from the group consisting of 5' -methylselenoadenosine, Se-adenosyl-L-homocysteine, gamma-glutamyl-methylselenocysteine, a compound of formula (I), and a compound of formula (III), wherein the effective amount alters glucose metabolism in hepatocytes or muscle cells as compared to cells not treated with the composition. In embodiments, the composition may exclude one or more of H (5' -methylthioadenosine), I (S-adenosyl-L-homocysteine) and/or J (γ -glutamyl-methyl-cysteine).

In an embodiment of the present application, there is provided the use of a composition comprising at least three different compounds selected from the group consisting of 5' -methylselenoadenosine, Se-adenosyl-L-homocysteine, γ -glutamyl-methylselenocysteine, a compound of formula (I) and a compound of formula (III) for treating diabetes in a subject.

In an embodiment, there is provided a method or use for inhibiting expression of G6pc in one or more hepatocytes, comprising: administering a composition comprising at least three compounds selected from the group consisting of 5' -methylselenoadenosine, Se-adenosyl-L-homocysteine, gamma-glutamyl-methylselenocysteine, a compound of formula I, a compound of formula II, a compound of formula III, and combinations thereof. In embodiments, the composition may exclude one or more of H (5' -methylthioadenosine), I (S-adenosyl-L-homocysteine), and/or J (γ -glutamyl-methyl-cysteine).

In an embodiment of the present application, there is provided the use of a composition comprising at least three different compounds selected from the group consisting of 5' -methylselenoadenosine, Se-adenosyl-L-homocysteine, γ -glutamyl-methylselenocysteine, a compound of formula (I) and a compound of formula (III) for inhibiting glucose-6-phosphatase.

In embodiments, the increase in FOXO phosphorylation or decrease in G6pc ranges from about a 50% increase or decrease to a 500% increase or decrease, respectively, as compared to a cell of the same type not treated with the compound. The magnitude of the response depends on the cell type, the particular compound, and the time of contact with the cell.

Selenium-containing compounds differentially affect gene expression in hepatocytes. In contrast to the effects that compounds and compositions have on hepatocytes as described herein, the same or similar compositions can affect neuronal cells in a reverse manner. For example, a composition as described herein reduces phosphorylation of FOXO3 and/or FOXO4 in a neuronal cell. Increased expression of PGC1a results in increased gluconeogenesis in neuronal cells. In contrast, the same compound or composition increased phosphorylation of FOXO3 and/or FOXO4 in hepatocytes, and expression of PGC1a was unchanged.

As described in the context of neuronal IMR-32 cells, PGC1a is a key gene for MT biogenesis and carbohydrate metabolism. In hepatocytes, consistent with FOXO, it is also used to drive transcription of genes involved in gluconeogenesis, but this drive cannot be performed in the absence of the nucleus of FOXO. We examined PGC1a protein expression and no significant changes in PGC protein levels in hepatocytes were observed by quantitative analysis after CDE combination treatment. However, due to the robust effect observed on FOXO phosphorylation in response to CDE, it is almost certain that it is excreted from the nucleus, so the level of PGC1a becomes less important as it requires FOXO to initiate the gluconeogenesis process. Taken together, these results indicate that the combination of CDEs does not affect PI3k/PDK1/AKT signaling and several other AKT direct or indirect downstream signaling molecules, in addition to the important FOXO described above. In other words, the combination of CDEs selectively inactivated FOXO in hepatocytes and this effect appeared to be independent of PI3K/PDK1/AKT signaling.

In embodiments, an effective amount of a compound and composition as described herein is an amount effective to inhibit expression of G6pc, modulate glucose metabolism, or increase FOXO phosphorylation in hepatocytes without toxicity to the cells. Gene expression in hepatocytes can be determined on samples obtained from subjects treated according to the compositions described herein using a number of assays. The effective amount is selected to be non-toxic to any of the exemplified cells including kidney cells, mouse skeletal cells, human neuronal cells, or mouse liver cells.

As is well known in the medical arts, the dosage for any one subject may depend on a number of factors, including the size of the patient, body surface area, age, the particular compound to be administered, sex, number and route of administration, general health, and interaction with other drugs being administered concurrently.

In embodiments, the effective amount of a selenium-containing composition of the invention administered to a subject to inhibit G6pc expression, modulate glucose metabolism, treat type II diabetes, or increase FOXO phosphorylation in liver or skeletal muscle cells is about 5 ug or more or 800 ug or less of a single desired bioactive selenium-containing compound of the invention or a plurality of desired bioactive selenium-containing compounds of the invention per day. When multiple desired bioactive selenium-containing compounds are present, the effective amount of the composition administered to the subject is the total amount of all desired bioactive selenium-containing compounds in the composition.

In embodiments, an effective amount of a biologically active selenium-containing compound of the invention to be administered to a subject in need thereof is from about 5ug to about 800 ug and each numerical value therein, from 5ug to about 700 ug and each numerical value therein, from 5ug to about 600 ug and each numerical value therein, from 5ug to about 500 ug and each numerical value therein, from 5ug to about 400 ug and each numerical value therein, from 5ug to about 300 ug and each numerical value therein, from 5ug to about 200ug and each numerical value therein, from 5ug to about 100 ug and each numerical value therein and from 5ug to 50 ug.

In embodiments of the invention, the composition comprising a compound described herein is preferably administered at 200ug or less per day or 50 ug or less per day. In embodiments of the invention, the dose may be adjusted according to efficacy or whether any significant signs of selenium poisoning such as garlic-flavoured respiration, hair loss or cuticle is observed in the subject.

In embodiments, in a composition comprising two or more compounds selected from the group consisting of 5' -methylselenoadenosine, Se-adenosyl-L-homocysteine, γ -glutamyl-methylselenocysteine, a compound of formula (I), a compound of formula (II), a compound of formula (III), and combinations thereof, the compounds are present in the composition in equal proportions. In other embodiments the ratio of one compound to another may be about 4:1 to 1: 1.

In embodiments, the dose is administered at least once daily for a period of time to achieve a steady state of elemental selenium in the blood. In embodiments, the dose is administered daily for at least 60 or 90 days. In embodiments, the dose is administered while the subject is experiencing symptoms of the disease or disorder. In embodiments, the subject is at an age where the risk of a mitochondrial-related disease is increased, e.g., at least 40 years of age.

In other embodiments, delivery to the liver can be targeted by making selenoglycosides or liver-targeting prodrugs by using liposomes or PLGA spheres with antibodies.

Other embodiments of the present application

In some embodiments, there is provided a composition consisting essentially of, or consisting of, a compound of formula (I):

R₂is H, acyl, alkyl, alkenyl, alkynyl, aralkyl, carboxy, cycloalkyl, C (O) R ', C (O) OR ', wherein R ' is selected from alkyl, cycloalkyl, aryl, aralkyl, OR heterocyclyl; or R ₁And R₂Together form a heterocyclic ring having 4-8 ring members and at least one heteroatom selected from oxygen or nitrogen;

R₄is H, acyl, alkyl, alkenyl, alkynyl, aralkyl, cycloalkyl, carboxyl or C-acylamino, or R₃And R₄Together with the atoms to which they are attached form a heterocyclic ring having 4-8 ring members and at least one heteroatom selected from oxygen or nitrogen;

R₅is oxo, hydroxy, alkyl, alkenyl, alkynyl, OR' OR absent; wherein R' is selected from alkyl, alkenyl, alkynyl, cycloalkyl, aryl or aralkyl;

R₆is oxo, hydroxy, alkyl, alkenyl, alkynyl, OR' OR absent; wherein R' is selected from alkyl, alkenyl, alkynyl, cycloalkyl, aryl or aralkyl;

R₇is H, alkyl, alkenyl, alkyneA group, ketone, amino alcohol, amino acid, OR ', Se-R', S-R ', wherein R' is selected from H, alkyl, cycloalkyl, aryl, aralkyl, OR heterocyclyl; and

R₈is hydrogen, azido, alkyl, alkenyl, alkynyl.

In a further embodiment, one or more of these compounds of formula (I) may be isolated and/or purified.

In some embodiments, there is provided a composition consisting essentially of or consisting of a compound of formula (I) or a pharmaceutically acceptable salt, hydrate, or prodrug thereof, wherein R is₁、R₃、R₄And R₈Each is H; r₂Is H, acyl, alkyl, alkenyl, alkynyl, aralkyl, carboxy, cycloalkyl, C (O) R ', C (O) OR ', wherein R ' is selected from alkyl, cycloalkyl, aryl, aralkyl, OR heterocyclyl; r₅And R₆Each is absent; and R₇Is an alkyl group or an amino acid; provided that 5 '-selenoadenosine methionine, dehydroxy 5' -methylselenoadenosine, ethylselenoadenosine, seleno (hydroxy) -selenophenyl- (3 '-deoxy-adenosine), allylselenoadenosine homocysteine, selenoadenosine-Se (methyl) -selenoxide, adenosine-hydroxyselenoxide, ethylselenoadenosine, seleno- (hydroxy) -selenophenyl- (3' -deoxy-adenosine), adenosine-hydroxyselenoxide, and selenoadenosine-Se (methyl) -selenoxide can each be excluded from the composition.

In a particular aspect, there is provided a composition consisting essentially of or consisting of a compound of formula (I): or a pharmaceutically acceptable salt, hydrate or prodrug thereof, which compound is 5' -methylselenoadenosine ("compound C").

In some embodiments, the composition consists essentially of or consists of a compound of formula (I) or a pharmaceutically acceptable salt, hydrate, or prodrug thereof, provided that 5 '-selenoadenosine methionine, dehydroxy 5' -methylselenoadenosine, ethylselenoadenosine, seleno (hydroxy) -selenophenyl- (3 '-deoxy-adenosine), allylselenoadenosine homocysteine, selenoadenosine-Se (methyl) -selenoxide, adenosine-hydroxyselenoxide, ethylselenoadenosine, seleno- (hydroxy) -selenophenyl- (3' -deoxy-adenosine), adenosine-hydroxyselenoxide, and selenoadenosine-Se (methyl) -selenoxide can each be excluded from the composition.

In some embodiments, there is provided a composition consisting essentially of, or consisting of, a compound of formula (II):

，

wherein R is₁Is H, acyl, alkyl, alkenyl, alkynyl, aralkyl, carboxyl, cycloalkyl, C (O) R ', C (O) OR ', wherein R ' is alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, OR heterocyclyl; or R ₁And R₂Together form a heterocyclic ring having 4-8 ring members and at least one heteroatom selected from oxygen or nitrogen;

R₃is H, acyl, alkyl, alkenyl, alkynyl, aralkyl, cycloalkyl, carboxyl or C-amido; or R₃And R₄Together with the atoms to which they are attached form a ring having 4-8 ringsA heterocycle of a member and at least one heteroatom selected from oxygen or nitrogen;

R₅is oxo, hydroxy, alkyl, alkenyl, alkynyl, OR' OR absent; wherein R' is selected from alkyl, alkenyl, alkynyl, cycloalkyl, aryl or aralkyl;

R₆is oxo, hydroxy, alkyl, alkenyl, alkynyl, OR' OR absent; wherein R' is selected from alkyl, alkenyl, alkynyl, cycloalkyl, aryl or aralkyl;

R₈Is hydrogen, azido, alkyl, alkenyl, alkynyl;

R₉is H, acyl, alkyl, alkenyl, alkynyl, aralkyl, carboxy, cycloalkyl, C (O) R ', C (O) OR ', wherein R ' is alkyl, cycloalkyl, aryl, aralkyl, OR heterocyclyl; or R₉And R₁₀Together form a heterocyclic ring having 4-8 ring members and at least one heteroatom selected from oxygen or nitrogen;

R₁₁is OH, OR, alkoxy, aralkoxy OR amino, wherein R is selected from alkyl, cycloalkyl, aryl, aralkyl, heterocyclyl OR a pharmaceutically acceptable salt OR inner salt.

In a further embodiment, one or more of these compounds of formula (II) may be isolated and/or purified.

In some embodiments, there is provided a composition consisting essentially of or consisting of a compound of formula (II) or a pharmaceutically acceptable salt, hydrate, or prodrug thereof, wherein R is₁、R₃、R₄、R₈And R₉Each is H; r₂Is H, acyl, alkyl, carboxyl, C (O) R ' OR C (O) OR ', wherein R ' is selected from alkyl, cycloalkyl, aryl, aralkyl, OR heterocyclyl; r ₅And R₆Is absent; r₁₀Is H, acyl, alkyl, alkenyl, alkynyl, aralkyl, carboxy, cycloalkyl, C (O) R ', C (O) OR ', wherein R ' is selected from alkyl, cycloalkyl, aryl, aralkyl, OR heterocyclyl; and R₁₁Is OH, OR, alkoxy, aralkoxy OR amino, wherein R is selected from alkyl, cycloalkyl, aryl, aralkyl, heterocyclyl OR a pharmaceutically acceptable salt OR inner salt.

In some embodiments, there is provided a composition consisting essentially of or consisting of a compound of formula (II) or a pharmaceutically acceptable salt, hydrate, or prodrug thereof, wherein R is₁、R₂、R₃、R₄、R₅、R₆、R₇、R₈、R₉、R₁₀As defined above and wherein R₁₁Is OH OR OR, wherein R is selected from methyl, ethyl, propyl, isopropyl, butyl, sec-butyl OR tert-butyl.

In a particular aspect, there is provided a composition consisting essentially of or consisting of a compound of formula (II) or a pharmaceutically acceptable salt, hydrate or prodrug thereof, said compound being 5' -selenoadenosine homocysteine (compound "D") or a pharmaceutically acceptable salt, hydrate or prodrug thereof.

In some embodiments, the composition consists essentially of, or consists of, a compound of formula (II) or a pharmaceutically acceptable salt, hydrate, or prodrug thereof; provided that 5' -selenoindenosine methionine, allylselenadenosylhomocysteine, selenadenosylhomocysteine and selenohydroxyadenosylhomocysteine can each be excluded from the composition.

In some embodiments, there is provided a composition consisting essentially of, or consisting of, a compound of formula (III):

wherein

R₃is OH, OR, alkoxy, aralkoxy OR amino, wherein R is selected from alkyl, cycloalkyl, aryl, aralkyl, heterocyclyl OR a pharmaceutically acceptable salt OR inner salt;

R₄is H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heterocyclyl or a pharmaceutically acceptable salt or inner salt;

R₅is oxo, hydroxy, alkyl, alkenyl, alkynyl, OR' OR absent; wherein R' is selected from alkyl, alkenyl, alkynyl, cycloalkyl, aryl or aralkyl;

R₆Is oxo, hydroxy, alkyl, alkenyl, alkynyl, OR' OR absent; wherein R' is selected from alkyl, alkenyl, alkynyl, cycloalkyl, aryl or aralkyl; and

R₇is H, alkyl, alkenyl, alkynyl, ketone, OR ', Se-R', S-R ', wherein R' is selected from H, alkyl, cycloalkyl, aryl, aralkyl OR heterocyclyl.

In some embodiments, there is provided a composition consisting essentially of or consisting of a compound of formula (III), or a pharmaceutically acceptable salt, hydrate, or prodrug thereof, wherein

R₁And R₂Each is H;

R₃is OH, OR, alkoxy, aralkyloxy ORAmino, wherein R is selected from alkyl, cycloalkyl, aryl, aralkyl, heterocyclyl, or a pharmaceutically acceptable salt or inner salt;

R₄is H or a pharmaceutically acceptable salt or inner salt;

R₅and R₆Is absent; and

R₇is alkyl, alkenyl or alkynyl.

In a further embodiment, one or more of these compounds of formula (III) may be isolated and/or purified.

In some embodiments, provided are compositions consisting essentially of or consisting of a compound of formula (III) or a pharmaceutically acceptable salt, hydrate, or prodrug thereof, wherein R is₁And R₂Each is H; r₃Is OH OR OR, wherein R is selected from methyl, ethyl, propyl, isopropyl, butyl, sec-butyl OR tert-butyl; r ₄Is H; r₅And R₆Is absent; and R₇Is an alkyl group which is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl or tert-butyl.

In some embodiments, there is provided a composition consisting essentially of or consisting of a compound of formula III, or a pharmaceutically acceptable salt, hydrate, or prodrug thereof, wherein R is₁And R₂Each is H; r₃Is OH OR OR, wherein R is selected from methyl, ethyl, propyl, isopropyl, butyl, sec-butyl OR tert-butyl; r₄Is H; r₅And R₆Is absent; and R₇Is methyl.

In a particular aspect, there is provided a composition consisting essentially of or consisting of a compound of formula (III) or a pharmaceutically acceptable salt, hydrate, or prodrug thereof, said compound being γ -glutamyl-methylseleno-cysteine ("compound E") or a pharmaceutically acceptable salt, hydrate, or prodrug thereof.

In some embodiments, the composition consists essentially of, or consists of, a compound of formula (III), or a pharmaceutically acceptable salt, hydrate, or prodrug thereof, provided that γ -glutamylselenocysteine- γ -glutamylcysteine, γ -glutamylcysteine-2, 3-DHP-selenocysteine, di- γ -glutamylselenocysteine, selenoglutazone- γ -glutamylcysteine, γ -glutamylselenocysteine- γ -glutamylcysteine, γ -glutamylcysteine-2, 3-DHP-selenocysteine, di- γ -glutamylselenocysteine and selenoglutazone- γ -glutamylcysteine can each be excluded from the composition.

In some embodiments, compositions are provided that consist essentially of or consist of one or more compounds according to one or more of formulas (I), (II), and/or (III), wherein each of the following compounds is excluded from the composition to minimize selenium toxicity, remove inactive or inhibitory compounds, and/or maximize the therapeutic index of the composition, wherein the excluded compounds are γ -glutamylselenocysteine- γ -glutamylcysteine, γ -glutamylcysteine-2, 3-DHP-selenocysteine, di- γ -glutamylselenocysteine, selenoglutatin- γ -glutamylcysteine, γ -glutamylselenocysteine- γ -glutamylcysteine, γ -glutamylcysteine- γ -glutamylcysteine, gamma-glutamylcysteine- γ -glutamylcysteine, and/or (III), Gamma-glutamylcysteine-2, 3-DHP-selenocysteine, di-gamma-glutamylselenocysteine, selenoglutaglutathione-gamma-glutamylcysteine, dehydroxy 5 '-methylselenoadenosine, ethylselenoadenosine, seleno (hydroxy) -selenophenyl- (3' -deoxy-adenosine), allylselenadenosylhomocysteine, selenoadenosine homocysteine, selenoylhydroxyadenosine homocysteine, selenoyladenosine-Se (methyl) -selenoxide, adenosine-hydroxyselenoxide, ethyl selenoyladenosine, seleno- (hydroxy) -selenophenyl- (3' -deoxy-adenosine), adenosine-hydroxyselenoxide, and selenoyladenosine-Se (methyl) -selenoxide.

In embodiments, any of the compounds described herein may be modified to extend half-life, protect the compound from oxidation, target the compound to a tissue, allow the compound to pass through the blood brain barrier, as described herein.

In some embodiments, there is provided a composition consisting essentially of, or consisting of, one or more compounds each according to formula (I). In some aspects, a composition comprising one or more compounds each according to formula (I) comprises 5' -methylselenoadenosine, or a pharmaceutically acceptable salt, hydrate, or prodrug thereof; and 5' -selenadenosylhomocysteine or a pharmaceutically acceptable salt, hydrate or prodrug thereof.

In some embodiments, compositions are provided that consist essentially of or consist of one or more compounds according to formula (I) and formula (III), respectively. In some aspects, a composition comprising one or more compounds according to each of formula (I) and formula (III) comprises 5' -methylselenoadenosine, or a pharmaceutically acceptable salt, hydrate, or prodrug thereof; 5' -selenadenosylhomocysteine or a pharmaceutically acceptable salt, hydrate or prodrug thereof; and gamma-glutamyl-methylseleno-cysteine or a pharmaceutically acceptable salt, hydrate or prodrug thereof.

In some embodiments, a composition consisting essentially of or consisting of one or more compounds according to each of formula (I) and formula (III) comprises 5' -methylselenoadenosine, or a pharmaceutically acceptable salt, hydrate, or prodrug thereof; and gamma-glutamyl-methylseleno-cysteine or a pharmaceutically acceptable salt, hydrate or prodrug thereof.

In some embodiments, there is provided a composition consisting essentially of, or consisting of, one or more compounds according to formula (II) and formula (III), respectively. In some aspects, a composition comprising one or more compounds according to each of formula (II) and formula (III) comprises 5' -selenadenosylhomocysteine or a pharmaceutically acceptable salt, hydrate or prodrug thereof; and gamma-glutamyl-methylseleno-cysteine or a pharmaceutically acceptable salt, hydrate or prodrug thereof.

According to another aspect, the present invention provides a pharmaceutical composition consisting essentially of or consisting of a therapeutically effective amount of one or more compounds of the present invention, or a pharmaceutically acceptable salt, ester or prodrug thereof, and a pharmaceutically acceptable diluent or carrier. 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) tragacanth powder; (5) malt; (6) gelatin; (7) talc powder; (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 glycerol, 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 hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) a ringer's solution; (19) ethanol; (20) a phosphate buffer solution; and (21) other non-toxic compatible materials used in pharmaceutical formulations.

The compositions may be formulated for any of the routes of administration described herein.

Example I

Synthesis and characterization of 5' -methylselenoadenosine ("C

The synthesis scheme and method are as follows:

sodium borohydride (227mg, 6.0mM in Ar^oBelow) was placed in a 200mL round bottom flask containing 20mL of absolute ethanol, equipped on a magnetic stirrer and placed in an ice cooling bath. Dimethyl diselenide (190uL, 376mg, 2.0mM) was added from syringe under cooling, stirring and Ar flow. After the pale yellow solution had changed color, solid 5 '-chloro-5' -deoxyadenosine (1,143g, 4.0mM) was added. 5-Cl-Ade is very insoluble in ethanol. An additional 100mL of ethanol was added to dissolve the precipitate. Stirring of the mixture was continued at room temperature for four days. MS was used to monitor conversion. After 5 days, a conversion of 75% is achieved. Evaporation of the solvent and collection of the product 3.22g (containing 20% SM) and purification by reverse phase (C-8) preparative chromatography gave 1.1g of pure product, the molecular weight of which was confirmed by mass spectrometry.

Example 2

Synthesis and characterization of Se-adenosyl-L-homocysteine ("D

The synthesis scheme and method are as follows:

5 '-chloro-5' -deoxyadenosine (639-62)

89G (0.366 mol, 1eq.) adenosine, 59.3ML (58G, 1.833 mol, 2eq.) anhydrous pyridine, and 1L anhydrous acetonitrile were placed in an oven-dried 2L 4-neck flask equipped with a dropping funnel, stirrer, gas inlet/outlet, and thermometer. The reaction apparatus was placed in an ice/salt bath. Starting stirring, and when the temperature of the solution drops below 3 ^oC, thionyl chloride was added very slowly (strong exotherm!). The temperature of the reaction mixture during the addition of thionyl chloride needs to be kept below 5^oC and continued for more than 4h (at this point the solution was yellow with a white-yellow precipitate at the bottom). The reaction was then left at ambient temperature overnight. The next morning, the majority of the precipitate was filtered off using a sintered glass filter and washed on the filter with 3 times a volume of 100ML of anhydrous acetonitrile, during which the precipitate became white in color. The wet precipitate was then transferred back to a 2L reaction flask containing a mixture of 800ML methanol and 160ML water, to which 80ML of concentrated ammonium hydroxide solution was added dropwise with mechanical stirring and cooling with a water bath. The mixture was stirred at ambient temperature for 45min and the white precipitate formed was separated from the liquid by vacuum filtration. The filtrate was concentrated to dryness using a vacuum rotary evaporator while the precipitate crystallized from-560 ML of hot water, cooled in an ice-water bath and the first crystals were filtered off and freeze-dried. This filtrate was then used as a solvent in the solid crystals produced by rotary evaporation from the first filtrate to give a second crop of product, which was also freeze-dried (2 days). Both batches of crystals were finally dried over phosphorus pentoxide in a vacuum desiccator for 2 days. 84G was obtained as white crystals in 80.5% yield. MS (286-M + H), mp. 187 ^oC。

Seleno adenosine homocysteine (655-40)

9.806G (50mM, 1eq.) of L-selenomethionine was charged to a 2L three-necked flask equipped with a thermometer, large cooling finger (with bubble-check on the exit), ammonia inlet (to the bottom of the flask) and magnetic stir bar and placed in a flask containing CO₂-a 2.5L double-layer vessel of an acetone cooling bath. Ar (Ar)^oPassing through a flask, then adding solid CO₂To an acetone bath and cooling hands. When the temperature in the flask drops below-35 deg.C^oAt C, anhydrous ammonia (gas) flow is started and when the liquid ammonia level reaches 800ML volume, gas flow is stopped. At this point a small piece of sodium metal was added to the well-stirred solution until the bluish-purple color of the solution remained for about 30 seconds. A total of 2.645G (115mM, 2.3 eq.) of sodium was added over 45 min. Stirring and cooling was maintained for another 30 min. All components are in solution at this point. 14.856G (52mM, 1.04 eq.) of anhydrous 5 '-chloro-5' -deoxyadenosine was added in a single portion and the reaction mixture was left overnight with stirring and very slow Ar^oAnd (4) streaming. The next morning (if all ammonia disappeared) 350ML of anhydrous methanol was added to the white solid present in the flask. The flask was placed in an oil bath, a reflux condenser was installed, and Ar was maintained^oGas flow and heating the oil bath to 50 deg.C ^oC continued for the subsequent 24 h. At this point the 1ML solution was acidified to pH 3.5 with a few drops of 0.1N HCl and the sample analyzed for the presence of substrate using mass spectrometry. If they are below 5%, the mixture can be acidified to pH 3.5 with 1N HCl, filtered from the salts, concentrated to dryness using a vacuum rotary evaporator and the crude product can be purified by crystallization from a water-ethanol mixture. The first selenadenosyl homocysteine crystals of 15.98G (74% yield) were 95% pure and could be used for biological studies without further purification.

Example 3

Synthesis and characterization of gamma-glutamyl-methylselenocysteine

The synthesis scheme and method are as follows:

synthesis of N-Boc- (O-tBu) -L-Glu-OSu (655-90)

N-Boc- (O-tBu) -L-Glu-OH (303mg, 1.0Mmol), N-hydroxysuccinimide (121mg, 1.05Mmol) and dicyclohexylcarbodiimide (227mg, 1.1Mmol) were suspended/dissolved in 15mL of anhydrous ether and 10uL of dimethylethylbenzylamine was added from a syringe to the reaction mixture. At ambient temperature (22)^oC) Stirring was maintained for 48 h. The mixture was filtered and the precipitate was washed 10 times with 10mL of diethyl ether. FiltrateConcentration and drying under high vacuum gave the product as white crystals (570mg, 90% yield). MS (M + Na)⁺) = 423.17。

Synthesis of N-Boc- (O-tBu) -L-Glu-MeSe-Cys-OH (655-90)

N-Boc- (O-tBu) -L-Glu-OSu (570mg, 0.9Mmol), methylselenocysteine (175mg, 0.8Mmol), triethylamine (152mg, 209. mu.L, 1.5Mmol) was added to a mixture of 6mL of 1, 4-dioxane and 2mL of water. The reaction mixture was magnetically stirred for 100 h. Thereafter, 1.21N HCl (1.65mL) was added, the reaction mixture was extracted with 3X 20mL of diethyl ether, and the extract was concentrated to dryness using a vacuum rotary evaporator to give 649mg of a waxy product which was subjected to preparative HPLC. 283mg of product were collected (75.6% yield). Mass spectrometry confirmed the molecular weight of the product and the presence of a single Se atom therein. Theory Ms C₁₈H₃₂N₂O₇Se = 468.42; 469.24M/e (M + H) was found⁺) And 491.24M/e (M + Na)⁺)。

Synthesis of gamma-glutamyl-methylselenocysteine (655-92)

A mixture of 283mg (0.6Mmol) of N-Boc- (O-tBu) -L-Glu-MeSe-Cys-OH, 2mL of thioanisole and 5mL of trifluoroacetic acid was stirred magnetically in an oil bath at 63 mL^oC heating for 6h and at ambient temperature (22)^oC) Left overnight. The reaction mixture was then added (dropwise) to vigorously stirred diethyl ether (20 mL). The precipitate formed was washed with 2 × 20mL of diethyl ether to give 138.3mg of a paste-like precipitate, which was then purified by preparative HPLC.

Example 4

The effects of three synthetic selenium organic compounds on mitochondrial function, cell survival and gene expression were tested in cell culture, alone or in combination. Cells tested included hepatocytes, kidney cells, neuronal cells, and skeletal muscle.

Materials and methods

Cell lines and AT compounds

Human embryonic kidney HEK293T cells were generously provided by doctor Qiatang Li (University of Louisville). Mouse skeletal muscle myoblast C2C12 cell line was gifted by doctor Xiao (University of Kentucky). Human neuroblasts IMR-32 cells and mouse liver cell line AML-12 were purchased from American type culture Collection (ATCC, Manassas, Virginia). All these cells were expanded in the culture medium suggested by the ATCC.

Compounds C (5 '-methylselenoadenosine), D (Se-adenosyl-L-homocysteine) and E (γ -glutamyl-methylseleno-cysteine) and their sulfur analogs H (5' -methylthioadenosine), I (S-adenosyl-L-homocysteine) and J (γ -glutamyl-methyl-cysteine) were identified as individual compounds of selenized yeast with less than 2% inorganic selenium and were obtained synthetically or from commercial sources (where appropriate). The purity of all tested compounds was verified to be ≧ 99% as determined by mass spectrometry.

Fluorescence analysis of mitochondrial potential in HEK293T cells

HEK293T cells were cultured on 8-chamber glass slides (1X 10)⁴Cells/chamber) for 24 hours, then treated (dissolved in sterile water) with various concentrations of the compound or its corresponding sulfur analog for 4.5 hr. According to the manufacturer's protocol, these compound-treated cells were rinsed twice with PBS and incubated with Mitotracker orange fluorescent dye (Invitrogen) at 37 ℃ for 30 min, then replaced with fresh PBS. The fluorescence signal (mitochondrial potential) of these living cells was then recorded using a Zeiss Axio vert.a1 fluorescence microscope (Thornwood, NY). At least three samples/treatment groups were analyzed under a fluorescence microscope.

Quantitative analysis of mitochondrial potential in C2C12, IMR-32 and AML-12 cells

For C2C12 cells, equal numbers of cells (1X 10)⁴) Inoculated into Corning 96-well clear bottom and black wall cell culture plates (VWR), cultured in 10% FBS DMEM medium for 24 hr, then treated with vehicle (sterile water), compounds C and D and their sulfur analogs for 24 and 48 hr. These vehicle-or compound-treated cells were rinsed twice with PBS, incubated with Mitotracker orange fluorescent dye at 37 ℃ for 30 min, and then replaced with PBS. The intensity of the Mitotracker orange fluorescence (mitochondrial potential) in these living cells was determined by a Bio-TeK Synergy HT Multi-Mode fluorescence microplate reader (Winooski, VT). The 8 samples/treatment were examined for the above analysis and the data are expressed as mean ± SEM of the 8 samples.

For AML-12 hepatocytes, cells were expanded in 10% FBS Dulbecco's modified Eagle Medium supplemented with 1X insulin/transferrin/selenium (ITS, Sigma) and Ham's F12 Medium (1:1) (DMEM/F12). The same number of cells (1X 10) was then added⁴Cells/well) were seeded in Corning 96-well clear bottom and black wall cell culture plates (VWR, Radnor, PA), cultured in ITS 10% FBS-free DMEM/F12 medium for 24 hr, and then treated with each compound or combination of compounds for 6, 24, and 48 hr. Mitochondrial potential in living cells was determined by quantitative analysis of fluorescence intensity using the Mitotracker orange dye described above. In addition, the above treated cells were also incubated with Hoechst 33342 dye (Invitrogen, Grand Island, NY) to stain the nucleus according to the manufacturer's protocol. The fluorescence intensity of nuclei stained by Hoechst 33342 dye was also measured by a fluorescent microplate reader. Mitochondrial potential/cell in living cells was obtained by normalizing the fluorescence intensity of Mitotracker orange dye to that of Hoechst 33342 dye in each well. The 8 samples/treatment were examined for the above analysis and the data are expressed as mean ± SEM of the 8 samples.

Similar to the above study on AML-12 cells, mitochondrial potentials/cells in live IMR-32 cells were determined using a Bio-TeK Synergy HT Multi-Mode fluorescent microplate reader with the following modifications. To improve the attachment of IMR-32 cells to the culture dish, the cell culture plates were pre-coated with 0.1% gelatin (Sigma, St. Louis, MO). 2X10⁴Cells/96-well were seeded in gelatinized 96-well plates for the above study. To reduce cell migration, Mitotracker orange and Hoechst 33342 fluorescent dyes (diluted in culture medium) were added directly to each well after vehicle-or compound-treatment. After dye incubation, the cell culture medium was carefully replaced with 1X PBS for quantitative analysis of fluorescence on a microplate reader. The 8 samples/treatment were checked for the above analysis and the experiment was repeated at least 5 times. Data are presented as mean ± SEM of 8 samples.

Cell viability assay

Cell viability of cultured cells Using the Promega's CellTiter96 waterborne One Solution Cell Proliferation Assay kit, manufactured according to manufacturerThe protocol of the quotient was determined. Briefly, equal amounts of C2C12 (1X 10) were added⁴Cells/well), AML-12 (1X 10)⁴Cells/well) and IMR-32 (2X 10)⁴Cells/well) were seeded on 96-well clarified plates (VWR) and treated with vehicle or compound for 24, 28 and/or 72 hr. Then, the cultured cells were incubated with AQueous One solution (100 ul/well) at 37 ℃ for 1 hr, and the absorbance at OD490 nm in each sample was measured by a Bio-Tek microplate reader. Cell viability of cultured cells was determined by subtracting the OD490 nm of the cultured cells from the OD490 nm of the medium alone (uninoculated cells). The 8 samples/treatment were examined for the above analysis. Data are presented as mean ± SEM of 8 samples.

RNA isolation and real-time PCR analysis

Human IMR-32 cells were seeded into gelatinized 6-well (6.5X 10)⁵Cell/well) or 24-well (1.3X 10)⁵Cell/well) plate, and mouse liver AML-12 cells in uncoated 6-well (3.33X 10)⁵Cells/well) or 24-well (6.7X 10)⁴Cells/well) plate. Cells were treated with vehicle (control) or each compound for 6, 24 or 48 hr. Total RNA was isolated from these cells using trizol (invitrogen) according to the manufacturer's protocol, followed by incubation with DNase I to remove any possible contaminating genomic DNA. RNA samples were then subjected to real-time PCR analysis using the Applied-Bioscience RT kit and pre-designed Taqman probe (Invitrogen) as previously described (Lan et al EMBO J2003). 3-6 samples were analyzed in each treatment group. Data were normalized by actin b (actb) or glyceraldehyde phosphate dehydrogenase (Gapdh) levels in each sample and expressed as mean ± SEM of 3-6 samples.

Protein preparation and western blot analysis

IMR-32 and AML12 cells were seeded in 6-well plates and then treated with vehicle and various compounds for 6 and 24 hr as described above. After treatment, cells were rinsed with ice-cold PBS and lysed on ice for 30 min in ice-cold RIPA buffer containing complete protease and phosphatase inhibitors (thermo-Fisher Scientific, Waltham, MA). Cell lysates were collected using cell scraper and pipette, then at 12000 g is inCentrifuging at 4 deg.C for 30 min to remove DNA precipitate, and obtaining protein extractA compound (I) is provided. Protein levels in supernatants of these cell lysates were determined using the Pierce Micro-BCA protein assay kit (the mo Scientific-Piece Biotechnology, Rockford, IL) according to the manufacturer's protocol.

For western blot analysis, 5 micrograms total protein from both the media-and compound-treated cells were subjected to SDS-PAGE gel separation and then transferred to a PVDF membrane as described previously (Reddy, Liu et al 2008 Science). Membranes were blocked in phosphate-buffered saline (PBS) containing 5% (w/v) bovine serum albumin (Sigma, St. Louis, MO) and incubated with a specific primary antibody, followed by HRP-conjugated anti-mouse or anti-rabbit secondary antibody (1:5000 dilution, Cell Signaling Inc.). All primary antibodies were purchased from Cell Signaling Inc, except G6PC (Santa Cruz), Actb (Li-COR, Lincoln, Nebraska), Elf2b epsilon, and pElf2b epsilon (Abcam, Cambridge, MA). Positive signals on the membrane blots were detected using Amersham's enhanced chemiluminescent Western blotting optimal Detection reagent (GE healthcare Life sciences, Pittsburgh, Pa.). Images of these luminescence signals on membrane blots were captured using the LI-COR Odyssey Fc Image system (Lincoln, Nebraska). The same membrane blots were cut into strips and re-blotted with another antibody as described in the GE WB ECL-optimal detection protocol (GE healthcare Life science, Pittsburgh, Pa.). Protein band densities on western blots were determined using either Li-COR Image studio software or NIH Image j software and then normalized by Actb levels in each sample. Data are presented as mean ± SEM of 3 samples per group.

Statistical analysis

Student's t-tests were performed to determine statistical differences between the two groups, as applicable. P values below 0.05 were considered significant.

Results and discussion: mitochondrial function

To test whether compound C or D can increase mitochondrial ("MT") potential in cultured human kidney cells, HEK293T cells were treated with 37.5 and 75ppb of compound C or D. These amounts are based on studies using the SeaHorse assay, where an effective dose of 100-150ppb was found. Compound C and its sulfur analog H were incubated with HEK293T cells for 4.5 hours and fluorescence analysis was performed under a microscope. As shown in fig. 1, a lower dose (37.5 ppb) of compound C increased the MT potential, while having less effect on the MT potential in renal cells treated with a higher dose (75ppb) of compound C for 4.5 hr. In contrast, all doses of compound D tested did not affect the MT potential (fig. 1).

Our results clearly demonstrate that compound C increases MT potential in kidney cells, whereas compound D has no effect. Considering that selenium is present in both compounds C and D, and that their respective sulfur analogs H and J proved unable to stimulate mitochondrial activity, it was concluded that the stimulatory effect of compound C in renal cells was due to the combined effect of selenium and the molecular structure surrounding selenium in this organoselenium compound. The stimulatory activity was not attributed to the selenium effect alone.

The importance of being able to increase mitochondrial activity in kidney cells can have very significant health benefits, especially in diabetic subjects. A recent study (Sharma et al J AM Soc Nephrol, 2013: 1901-12) concluded from metabolic analysis of urine from diabetic populations that diabetic nephropathy is clearly associated with decreased mitochondrial function. Diabetic nephropathy (DKD) is the major cause of end-stage renal disease, and the authors concluded that therapeutic approaches to restore or increase mitochondrial function could improve or even prevent DKD. An additional interest lies in the fact that the authors linked mitochondrial dysfunction in these cases to a transcriptional co-activator called PGC1 a. As discussed later, PGC1a is the target of the synthetic selenium compounds described in the present application.

Compounds C and D increase mitochondrial potential in mouse C2C12 cells

To test whether compounds C and D affect MT potential in skeletal muscle cells, C2C12 cells were treated with vehicle (water, control) or various concentrations of selenium and sulfur compounds, and then subjected to quantitative analysis of MT potential. As shown in fig. 2, MT potential decreased after 24 hr treatment with sulfur analogs in C2C12 cells (compared to control), indicating that sulfur compounds showed some MT toxicity in skeletal muscle cells. In contrast, selenium compounds C and D significantly increased the MT potential (fig. 2).

Two aspects of this result are particularly unexpected and unexpected. First, compound D, although inactive in kidney cells, is highly effective in stimulating mitochondrial activity in muscle cells (approximately 2-fold the control level at its most effective dose). Furthermore, at lower doses (37.5 and 75 ppb, fig. 2), it proved to be more effective than compound C, which (as shown above) was the only test compound effective in renal cells.

Second, the sulfur analogs of C and D, not only had no effect relative to vehicle-treated control cells, but were inhibitory (in some cases reduced 2/3) to mitochondrial activity. This effect has not been reported in the past, but may help explain the cause of the pre-diabetic effect in subjects receiving these supplements in some studies using selenium enriched yeast preparations. Although selenium replaces the sulphur in the normal sulphur-containing molecules of yeast during selenium enrichment, there are always sulphur-containing molecules with certain advantages in the final preparation (sulphur is a major element and cannot be completely removed from any growth or fermentation process). Thus, the presence of certain sulfur-containing molecules in selenium-enriched yeast may, in some cases, inhibit mitochondrial activity and lead over time to pre-diabetic states. It is well documented in the literature that adult-onset diabetes is associated with a gradual decrease in mitochondrial activity over a period of years. This is particularly important in the case of skeletal muscle, which is estimated to utilize 75-80% of the daily glucose intake. Even a modest reduction in the ability of muscle mitochondria to effectively utilize glucose can lead to serious health problems over time. For example, noting a 2-fold stimulation of mitochondrial activity in C2C12 muscle cells in response to compound D may represent a way to avoid or delay mitochondrial decline in muscle tissue in pre-diabetic or diabetic subjects.

To investigate whether the observed increase in MT potential due to compounds C and D was due solely to an increase in viable cell number, we performed a cell viability assay. It was found that compounds C and D did not affect cell viability of C2C12 cells after 24 hr and 48 hr treatment with the same dose of these compounds (data not shown). In summary, our results show that compounds C and D have no negative effect on cell survival, but can transiently increase MT potential in C2C12 cells.

Compounds C, D and E transiently increased MT potential in IMR-32 cells

To investigate whether selenium compounds can modulate MT function in neuronal cells, human IMR32 cells were treated with various compounds for 6, 24, and 48 hr, followed by mitochondrial potentiometry. As shown in figure 3 (upper panel), compound D or E or a combination of both significantly increased MT potential for 6 hr compared to control normal cells (treated with aqueous vehicle), while a trend of increased MT potential was also observed in compound C-treated IMR-32 cells. An increase in MT potential was also observed in cells treated with compounds H or I and J (compare to control, upper panel of fig. 3). However, the MT potential increase was more pronounced in cells treated with compound D or E or DE combination than their corresponding sulfur analogs (upper panel of fig. 3).

A significant increase in MT potential was observed in all selenium compound-treated cells 24 hours after compound treatment (compared to control). In general, the MT potential increase was more pronounced in cells treated with the combination of selenium compound C, D, E or CDE compared to the combination of their sulfur analogs H, I, J or HIJ. It should be noted that in one instance, the combination of I and J produced the highest mitochondrial response at the 24 hr time point. However, at 48 hr, there was no significant change in MT potential in all tested groups. Taken together, these results indicate that selenium compound C, D, E or a combination of these compounds can transiently increase MT potential in IMR-32 cells and that the effects of these selenium compounds are more pronounced than their sulfur analogs.

Again, there are clearly significant cell-specific and compound-specific responses in this experiment, which underscores the different responses of cell types to these specific selenium compounds and their combinations. Furthermore, rather than being inhibitory to mitochondrial activity as in the case of myocytes, sulfur compounds stimulate mitochondrial activity in IMR-32 cells-although generally to a lesser extent than selenium compounds. The transient or temporary effect observed in this case indicates that these compounds or their combinations must be repeatedly administered to the treated subject in order to maintain the effect on mitochondrial activity; essentially daily doses may be required.

MT potential increase by repeated D or E treatment in IMR-32 cells for a total of 48 hours

The transient effects of compounds C, D and E on MT potential observed above suggest that we tested whether repeated treatments could increase MT potential. Thus, IMR-32 cells were first treated with compound C, D or E for 24 hr, then treated again with freshly prepared compound C, D or E for another 24 hr. As a negative control, IMR-32 cells were treated with compound C, D or E only once for 48 hr. Then, MT potential measurement was performed on these cells. As expected, compound C, D or E extended single treatment for 48 hr (continued for 48 hr single treatment) did not affect MT potential in IMR-32 (upper panel of fig. 4). However, repeated treatments of compound D (both 15 and 150 ppb) or E (dose 150 ppb) significantly increased the MT potential (compared to control or sulfur analogs I or J).

Compound C, D, E has no toxic effects on cell survival of IMR-32 cells

To test whether there were any toxic effects of selenium compounds on the survival of IMR-32 cells, cell viability assays were performed on cells after 24, 48 and 72 hours of treatment with each compound. As shown in fig. 5, treatment with all compounds did not result in a significant decrease in viable cells in IMR-32 for the time points tested. In fact, there was a small but significant increase in cell viability in the cells after 48 and 72 hours of selenium compound treatment (upper and lower panels in fig. 5). These data indicate that selenium compounds have no toxic effect on the survival of IMR-32 cells, but rather have a small but significant beneficial effect on the survival of neuronal cells. It should be noted, however, that any observed increase in cell viability in this experiment was too minimal to account for the increase in MT potential observed in the IMR-32 cell line in response to certain selenium compounds or their analogs.

Experimental data confirm the ability of selected synthetic selenium compounds to increase or decrease mitochondrial activity in mitochondria isolated from rat brain

The water soluble extract is obtained from selenized yeast. The water soluble extract comprises up to 25% of the total selenium present in the preparation. We reasoned that these selenium species were first released/digested from selenized yeast as it passed through the intestinal tract. After identifying selenium-containing compounds in the extract by mass spectrometry, we synthesized a number of selenium-containing compounds and peptides. Thus, this round of synthesis and purification yielded a panel of 9 selenium-containing species for further testing. Given the small amount of material thus produced (low milligram amounts), it was considered impractical to conduct feeding studies in live animals. Since we are mainly concerned with the possible effect of these selenium species on mitochondrial bioenergy, it was decided to test these selenium molecules directly using mitochondria.

The concentration ranges low (50ppb), medium (500 ppb) and high (1ppm) selenium were initially tested as possible ranges of compounds. Based on the fact that no toxicity to mitochondria was observed in the mid-range, we chose a 500ppb (5uM) concentration for our compound screening, using mitochondrial bioenergy as the primary outcome measure. Adult rat brain ficoll purified mitochondria were incubated with 9 compounds for 30 min at 37 ℃ and then loaded in triplicate into a Seahorse Biosciences flow analyzer. We measured OCR (oxygen consumption rate) parameters at three respiratory phases, including ATP synthesis (phase III), complex I-dependent (NADH-driven) maximum respiratory capacity (phase V) _FCCP) And complex II (FADH-driven) dependent maximum respiratory capacity (stage V)_succ)。

The results show that different selenium compounds derived from water extracts have different activities in being able to increase mitochondrial potential.

Compound 5: valine-selenomethionine-arginine

Compound 6: leucine-valine-selenomethionine-arginine

Compound 7: leucine-threonine-glycine-selenomethionine-alanine-phenylalanine-arginine

Compound 8: seleno glutathione dimer

Compound 9: methylselenoadenosine

Compound 10: glutamyl selenocysteine

Compound 25: total aqueous extract of yeast, pH 6.0

Compound 28: oxidized glutathione

Compound 30: glutamyl cysteine

TABLE 1

Specifically, compounds 6 and 9 increase mitochondrial potential, while compounds 7, 10 and 25 inhibit mitochondrial potential. These results indicate that compounds derived from aqueous extracts of selenized yeast have very different effects on mitochondrial potential, which has led us to isolate, screen and synthesize candidate compounds with the aim of selecting only those compounds that give a positive response to biological processes.

Experimental data confirm the ability of the synthesized selenium compounds to restore stressed mitochondria isolated from rat brain to normal activity

The following examples describe experiments performed on a SeaHorse external flow analyzer; the analyzer is an instrument for measuring the mitochondrial respiration rate expressed as oxygen consumption rate or OCR. The experiment used mitochondria from the cortex of normal rats maintained with normal experimental feed, which was not fortified with any additional selenium source. Figure 6 shows a respiration map (upper line) of normal mitochondria, with final OCR being the measured distance between the end of the map line and the X-axis.

Identical samples of the same mitochondrial preparation were treated in exactly the same manner as the control samples except that calcium (10 micromolar final concentration) was added to stress or damage the mitochondria by depolarizing them. As shown (lower line), OCR dropped from a value of 1,677 to 1,066 picomoles/min O₂。

Again, the same rat brain cortical mitochondrial samples were incubated as described above except that they contained calcium (10 μ M) and 150 ppb of compounds D and E, respectively. C was not tested. It is evident that OCR of compound-treated calcium-stressed mitochondria was restored to near control levels, i.e. 1,564 pmol/min O2 in the case of compound D, and 1,531 pmol/min O2 in the case of compound E.

From this result and those using repeated experiments with rat brain mitochondria, we conclude thatSelenium compounds not only have the ability to increase the mitochondrial activity of normal mitochondria (see examples using various types of unstressed cells), but also can be usedRecoveryThe respiratory capacity of stressed or damaged mitochondria.

MT potential was elevated in mouse hepatic AML-12 cells by a combination of C, D and E, but not by the compounds alone

To investigate whether selenium compounds could modulate MT function in hepatocytes, mouse AML-12 cells were treated with various compounds for 6 and 24 hr, followed by mitochondrial potential assays. As shown in figure 7, treatment with 150 ppb of compound C, D or E or its corresponding sulfur analog alone did not significantly affect the MT potential compared to control normal cells (treated with aqueous vehicle). However, the combination of C, D and E resulted in a highly significant increase in MT potential compared to both vehicle-or HIJ-treated groups (fig. 7). Thus, in hepatocytes, the specific combination of compounds C, D and E was required to cause an increase in MT potential, while the individual compounds and their sulfur analogs had no effect. This effect is quite different from that observed in neuronal cells and has not previously been demonstrated in the literature.

Down-regulated UCP2 expression in AML-12 cells by a combination of compounds C, D and E

One candidate gene family with the ability to increase or decrease mitochondrial activity is the so-called uncoupling protein gene or UCP. The increase in MT potential observed in response to treatment with the combination of CDEs allowed us to test whether these genes are differentially regulated in vehicle-, HIJ-, and CDE-treated AML-12 cells. In normal AML-12 cells, Ucp2 mRNA expression levels were 416-fold higher than Ucp1, whereas Ucp3 mRNA was not detected by real-time RT-PCR (FIG. 8A, data Ucp3 not shown). Treatment of AML-12 cells with CDE compounds did not affect Ucp1 expression (FIG. 8B). However, there was a significant reduction in Ucp2 mRNA expression in AML-12 cells after treatment with CDE, but not HIJ (fig. 8C). Because Ucp2 inhibits MT potential, a decrease in Ucp2 expression may be at least partially responsible for the increased MT potential in AML-12 cells caused by the CDE compound. Because obese patients often have high levels of Ucp2 in the liver, reduced expression of Ucp2 by CDE indicates that CDE compounds may be beneficial for treating obesity.

The compound has no toxic effect on survival of AML-12 cells

To test whether the selenium compounds had any toxic effect on the survival of AML-12 cells, cell viability assays were performed on the cells after 48 hr of treatment with each compound. We found that no treatment (both single compounds and their combinations) resulted in a significant reduction in cell viability in AML-12 cells (fig. 9), confirming that the selenium compound had no toxic effect on the survival of AML-12 cells.

Combination of Compounds C, D and E Down-regulated UCP2/3 in IMR-32 cells

As previously described, UCPs such as Ucp1, 2 and 3 are key genes that regulate MT potential. The increase in MT potential observed in IMR-32 cells in response to CDE (fig. 3, top two panels) suggests that we test whether there is differential expression of these three genes in response to combination treatment. Ucpl mRNA was found to be undetectable by real-time RT-PCR analysis in normal IMR-32 cells (data not shown). Treatment of IMR-32 cells with CDE compounds, but not HIJ, for 6 hours resulted in a significant reduction in expression of both Ucp2 and Ucp3 (fig. 10A-B). Ucp2 expression was significantly reduced in both the HIJ-and CDE-treated groups (fig. 10A), while a significant reduction in Ucp3 expression was also observed in CDE-treated IMR-32 cells (fig. 10B) at 24 hours treatment. These results indicate that Ucp2/3 down-regulation may be at least one cause of the increase in MT potential observed in IMR-32 cells in response to CDE compounds.

Discussion of the related Art

Thus, we have provided evidence that three synthetic organoselenium compounds, alone or in various combinations, have the ability to significantly increase mitochondrial activity in different cell types; i.e., kidney cells, skeletal muscle cells, neuronal cells, and liver cells. Mechanistically, we note that regulation of UCP may provide an explanation for this increase, and evidence is provided below that expression of other proteins important for mitochondrial function and biogenesis may also be favorably affected by these compounds. However, regardless of the mechanism of action, the fact that these compounds can stimulate mitochondrial activity in a trans-tissue manner means that they may be particularly valuable in ameliorating the development and progression of seemingly diverse diseases, such as Alzheimer's Disease (AD) and type 2 diabetes (T2 DM).

In the case of alzheimer's disease, the rapidly increasing literature supports the idea that AD originates from impaired glucose input in the brain, a lack of energy metabolism, mitochondrial dysfunction, chronic oxidative stress and DNA damage (reviewed in de la Monte and Wands, 2008). For example, many studies have concluded that insulin deficiency and insulin resistance can mediate many of the effects observed in AD-degeneration. Furthermore, T2DM has been found to cause brain insulin resistance, oxidative stress and cognitive impairment. Extensive disturbances in the brain insulin and insulin-like growth factor (IGF) -signaling mechanisms may explain many of the molecular, biochemical, and histopathological effects seen in AD.

Well-known statistics published by the american society for alzheimer's disease indicate that approximately 80% of AD patients have T2DM at the same time. These and other reasons have led many researchers to use the term "type 3 diabetes" to reflect the fact that AD represents a form of diabetes that selectively affects the brain and has molecular and biochemical features that overlap with both type 1 and type 2 diabetes.

The link between AD and type 2 diabetes is becoming very reliable in the scientific literature, but it is the link between these diseases and mitochondrial dysfunction that is the case It is T2DM, which combines a deficiency in insulin secretion by the pancreas with insulin resistance in peripheral tissues, particularly the liver and skeletal muscle, caused by a modest and gradual loss of mitochondrial respiratory function over a long period of time (Lowell and Shulman, 2005). Thus, any agent that can increase mitochondrial function in different tissues may be of great value as an intervention to track conditions that arise from decreased mitochondria.

Results and discussion of Alzheimer's disease onset

Combination of compounds C, D and E downregulating PSEN in IMR-32 cells

IMR-32 cells have been reported as a suitable in vitro model system for studying the onset of Alzheimer's Disease (AD) (Neill et al J. Neuroscience Res. 199439: 482). One of the important pathological features of AD is amyloid plaques, which occur between neurons and which promote brain atrophy and cell death. The mechanisms involved in amyloid plaque production are complex, but rely primarily on the action of an enzyme called β -secretase (BACE), which acts in concert with a multi-enzyme complex called γ -secretase. In summary, these enzymes function to aberrantly process brain proteins called Amyloid Precursor Protein (APP) in AD. The resulting product is an abnormal amyloid beta peptide, which aggregates together to form plaques.

As noted, γ -secretase is actually a complex composed of four known members: presenilin-1 (PSEN1 or PSEN), Nicastrin, APH-1 (antioxidant Pharynx Defect 1) and PEN2 (presenilin enhancer 2). Other orthologs of PSEN are presenilin 2 or PSEN 2. Although it is important that all four components function properly for gamma-secretase, in particular, two components have been the focus of the pharmaceutical treatment of pipeline. These fractions are presenilin 1 and Nicastrin. This is because presenilin 1 is the actual catalytic component of gamma-secretase-this component physically cleaves amyloid precursor protein. Furthermore, the gene of presenilin 1 is the gene that is most frequently mutated in familial AD. Compared with PSEN2, PSEN1 is more abundant and better determined in function. There is interest in Nicastrin not because it is catalytic, but because it binds and orients APP so that presenilins can cleave it. Thus, PSEN1 and Nicastrin are the most interesting targets for AD intervention focused on γ -secretase.

It should also be noted that several groups of studies suggest that AD results from a gradual decline in mitochondrial function as people age. In this regard, it is also of interest to note that one of the measurable preferential physiological changes during AD disease is insufficient uptake and utilization of glucose by brain cells; it is evident that these two phenomena may be correlated. Thus, we examined the expression of the gene encoding PSEN in IMR32 cells treated with a combination of compounds C, D and E (CDE) and a combination of their sulfur analogs (HIJ).

We found that PSEN expression in normal IMR32 cells was almost 8-fold higher than PSEN2 (fig. 10C). More importantly, PSEN1 expression was significantly reduced in CDE-treated cells compared to control or HIJ groups (fig. 10D). In contrast, there was no significant change in PSEN2 expression between the control, HIJ and CDE groups (fig. 10E). These results indicate that selenium compounds can selectively down-regulate PSEN expression in IMR-32 cells instead of PSEN2, and that a leader for amyloid plaque formation may be present in CDE compounds.

Compound C, a lead compound for preventing cleavage of APP for plaque formation in AD by targeting the gamma-secretase complex genes PSEN and Nicastrin in IMR-32 cells

As described above (fig. 10D), the compound CDE mixture inhibited PSEN expression, indicating the presence of biological leads against the gamma-secretase component for the production of amyloid β peptide in these three compounds. This study was limited to PSEN expression, but since gamma secretase is a multienzyme complex, another important component protein, Nicastrin, is included in this particular example. To identify lead compounds, IMR-32 cells were treated with individual compounds and subjected to Western blotting and RT-PCR analysis.

As shown in FIG. 11A, PSEN and Nicastrin, but not PEN2 and the β -secretase BACE protein, were attenuated in IMR-32 cells after 24 hr treatment with Compound C. Quantitative analysis showed that only C caused a significant decrease in both PSEN and Nicastrin protein levels, while D and E compounds also caused a trend towards decreased expression of Nicastrin protein (fig. 11B-C). Consistent with the attenuation of PSEN expression, compound C significantly reduced PSEN mRNA expression not only at 6 hr but also at 24 hr of treatment (fig. 11D-E). Similarly, compound C treatment also resulted in a trend toward decreased Nicastrin mRNA expression in IMR-32 cells after 6 hr treatment (fig. 11F), and more importantly, significantly decreased Nicastrin mRNA expression in IMR-32 cells after 24 hr treatment (fig. 11G). In contrast, compound D and E treatment did not inhibit, but stimulated PSEN and Nicastrin mRNA expression in IMR-32 cells (fig. 11E and 11G).

In summary, our data indicate that compound C can inhibit the expression of both PSEN and Nicastrin at both mRNA and protein levels. These results indicate that compound C is an anti-AD compound among the three candidates and is achieved by targeting gamma-secretase complex components known to cause plaque formation in AD, more particularly PSEN and Nicastrin.

Compound C, a cell-specific GSK3b down-regulator in IMR-32 cells for Tau protein hyperphosphorylation

In addition to amyloid plaques, the second major pathology in AD is known as neurofibrillary tangles, NFTs, or simply tangles. It is well characterized that tangle formation in AD is caused by hyperphosphorylation of a protein called Tau and that this phosphorylation is caused by kinases such as DYRK1A (bispecific tyrosine-phosphorylation-regulated kinase 1A) and mainly Gsk3b (glycogen synthase kinase 3 β). To investigate whether selenium compounds C, D or E are likely to promote a reduction in tangle formation in AD, we first investigated the phosphorylation status of two AD markers pTau S396 and pTauS400/T403/S404, as well as the total Tau protein concentration in IMR32 cells. Phosphorylation of Tau at a designated site is associated with destabilization and eventual tangle formation of Tau protein. For this purpose, cells were treated with compounds C, D and E for 6 and 24 hr, followed by Western blot analysis.

As shown in fig. 12A, the protein levels of all Tau protein species tested were not affected at the 6 hr treatment. However, after 24 hr treatment, protein levels of pTauS396, pTauS400/T403/S404 and/or total Tau in IMR-32 cells were significantly down-regulated by compounds C and/or E. Quantitative analysis indicated that compound C did not affect total Tau protein levels, but significantly inhibited phosphorylation of Tau on S400/T403/S404 but not on S396 (fig. 12B-C). Compound D had no effect on Tau phosphorylation on all tested serine/threonine residues or on total Tau protein levels, while Tau phosphorylation on S396 and S400/T403/S404 and total Tau protein were significantly down-regulated by compound E (fig. 12B-C).

Analysis of the ratio of total pTauS396 and pTauS400/T403/S404 to total Tau protein showed that only compound C, but not D or E, significantly attenuated total phosphorylation of Tau protein in IMR-32, even with the tendency of compound E to cause reduced Tau phosphorylation on all serine/threonine residues tested (fig. 12E).

In summary, our data show that compound C can significantly inhibit Tau phosphorylation in IMR32 cells, but does not affect the total Tau protein, while compound D does not have any effect in this process. Compound E may also have the effect of modulating Tau phosphorylation, but the effect of this compound may be through down-regulation of total Tau protein in IMR-32 cells. Given that hyperphosphorylation of Tau is responsible for entanglement formation in AD, our data suggests that C inhibits hyperphosphorylation of Tau, which promotes entanglement formation in AD.

To investigate whether compound C down-regulated Tau phosphorylation was due to Gsk3 and DYRK1A (two key kinases for Tau phosphorylation in AD), western blot analysis was performed to examine their protein levels. As shown in fig. 12A, phosphorylation of Gsk3b, total Gsk3a, and DYRK1A proteins were not affected by any of the three compounds in IMR-32 cells. However, Gsk3b protein levels were significantly reduced in IMR-32 cells after 24 hr, not 6 hr, treatment with compound C, not D or E (fig. 12A). Quantitative analysis showed a statistically significant reduction in Gsk3b protein levels in compound C, but not D or E treated IMR-32 cells (compared to control cells) (fig. 12F).

To further confirm that Gsk3b expression was inhibited by compound C, quantitative RT-PCR was performed to check its mRNA levels. As shown in fig. 12G, Gsk3b mRNA levels were significantly reduced in IMR32 cells after 6 hr treatment with compound C. A trend towards reduced Gsk3b mRNA expression was also observed in IMR-32 cells after 24 hr treatment with Compound C (FIG. 12H). In contrast, compound D and E treatment did not significantly inhibit Gsk3b mRNA expression in IMR-32 cells for 6 hr (fig. 12G). Instead, there was a significant increase in Gsk3b mRNA expression in IMR-32 cells after 24 hr treatment with compound D or E (fig. 12H). Taken together, these data indicate that compound C can inhibit Gsk3b expression at both mRNA and protein levels, and that down-regulation of Gsk3b by compound C may be responsible for the reduced Tau phosphorylation described above.

To investigate whether the inhibitory effect of compound C on Gsk3b expression was neuronal cell-specific, we examined the protein levels in mouse hepatocytes. Protein extracts were prepared from hepatocytes simultaneously and under identical experimental conditions as described above for IMR-32 cells. Gsk3b protein levels in hepatocytes were found to be unaffected by compound C (fig. 16). Therefore, down-regulation of Gsk3b expression is neuronal cell-specific. From a therapeutic perspective, the lack of response of GSK3b to compound C in hepatocytes may be of significant value. This means that compound C can be used as a therapeutic agent in neuronal tissue without risking severe interference with hepatic glucose metabolism.

Taken together, our results indicate that compound C is a neuronal cell-specific Gsk3b down-regulator and can inhibit Tau phosphorylation in neuronal cells, which would be beneficial for tangle formation in AD. These data further provide in vitro evidence that compound C may be a valuable therapeutic agent in the field of AD.

CDE causes neuronal-specific down-regulation of FOXO phosphorylation and up-regulation of PGC1a protein in IMR32 cells

In view of the effect of CDE compounds on mitochondrial activity observed in IMR-32 cells, we wanted to examine whether these compounds could modulate important factors responsible for cell growth and metabolism, mitochondrial function and energy. Foxo (forkhead box) proteins are an important family of nuclear transcription factors with diverse roles in cell proliferation, differentiation and survival. They control, in part, key functions of the cell, such as gluconeogenesis (production of glucose from non-sugar substrates). Their entry into the nucleus is controlled by phosphorylation, phosphorylated FOXO is excluded from the nucleus, and dephosphorylated FOXO is accessible.

PGC1a (peroxisome proliferator-activated receptor gamma coactivator 1- α) is a potent transcriptional activator that regulates genes involved in energy metabolism, and is also a major regulator of mitochondrial biogenesis and growth. It provides a direct link between external stimuli (e.g. movement) and the regulation of mitochondrial biogenesis. It performs different functions by co-activating genes in combination with different transcription factors. In the context of neuron-specific mitochondrial activity and AD progression, it is interesting to note that PGC1a expression decreases with dementia progression in the brain of Alzheimer's disease (Qin et al 2009. Arch Neurol; 66: 352-.

Therefore, we were interested in investigating whether any of the CDE compounds could affect the expression of these potent signalling factors. As shown in FIG. 13, phosphorylation of FOXO4 and FOXO3 was down-regulated after 6 and 24 hr CDE treatment in IMR-32 cells, while PGC1a protein was increased after 24 hr treatment in IMR-32 cells. There was little or no effect on protein expression of other tested signal transduction molecules. Increased PGC1a protein may promote MT biogenesis, which may provide another explanation for the reason MT potential is increased by CDE combinations in IMR-32 cells (fig. 3, top and middle panels). It was well characterized that dephosphorylation of FOXO such as FOXO4 at T28 and S193 resulted in nuclear localization of the FOXO protein. This result strongly suggests that the combination of CDE compounds is likely to enhance the nuclear FOXO effect. Taken together, our data indicate that CDE comprises one or more FOXO activators and positive PGC1a modulators in neuronal cells.

This result may be very meaningful from a therapeutic perspective in neuronal cells such as the brain. It is well established that co-localization of certain FOXO proteins and PGC1a in the nucleus can lead to efficient activation of gluconeogenesis (glucose production). Although such a situation may not be desirable in the liver of diabetic subjects, it may be considered very advantageous in the case of early AD subjects, where glucose input to neurons is impaired and thus brain cells lack their main energy source. In such a case, an increased ability to produce glucose from the carbon backbone of other molecules would be highly advantageous.

To test whether the observed FOXO and PGC1a activation (described above) was neuronal cell-specific, we also examined their protein levels in mouse hepatic AML-12 cells. Protein extracts were prepared from hepatocytes simultaneously and under identical experimental conditions as IMR-32 cells. However, the effect observed in hepatocytes (fig. 15), described in the latter section, was quite opposite to that obtained for IMR-32 cells, and certainly suggests that FOXO dephosphorylation and PGC1a upregulation by CDE are not tissue-wide phenomena and may be restricted to neuronal cells.

Compound C, a neuron-specific FOXO activator and PGC1a up-regulator

As described above, the CDE compounds together led to FOXO dephosphorylation and stimulation of PGC1a protein expression (fig. 13). To identify which compounds in the CDE mixture had these effects, we examined the expression of the aforementioned proteins in C-, D-and E-treated IMR32 cells. As shown in fig. 14A, pFOXO4 levels were significantly reduced after 6 hr treatment of all three compounds in IMR32 cells. A decrease in pFOXO 4T 28 levels was also observed in C-and E-but not D-treated IMR-32 cells after 24 hr treatment. Quantitative analysis indicated that C, D and E treatment for 6 hr and C and E treatment for 24 hr resulted in a statistically significant reduction in pFOXO4 levels in IMR-32 cells (FIGS. 14B-C).

It is important to note that neither CDE combination nor C, D or E treatment alone resulted in any change in the total FOXO level (fig. 13 and 14), but only altered the control of the FOXO effect by dephosphorylating it. It is unlikely that the decreased FOXO phosphorylation was attributed to down-regulation of AKT phosphorylation, as pAKT protein levels were not significantly decreased in C-, D-or E-treated cells (fig. 14A). Furthermore, pElf2beS539 protein levels were also unaffected by C, D or E, indicating that these compounds likely did not affect protein translation in neuronal cells (fig. 14A). In contrast, PGC1a protein levels were significantly increased after 24 hr treatment for all three compounds (fig. 14A, 14D), which may explain why MT potentials were upregulated by these compounds in IMR-32 cells (fig. 3, top and middle panels). Taken together, our data indicate that compounds C and E are FOXO activators and PGC1a up-regulators, while compound D is PGC1a up-regulator and has a mixed effect in FOXO phosphorylation modulation.

Discussion of the related Art

When considered collectively, our results in neuronal cells use compound C as a beneficial agent for eliciting beneficial effects in these cells. Although the results described directly above indicate that all three compounds are capable of causing FOXO dephosphorylation and PGC1a up-regulation-effects that are highly desirable from a neuronal cell energy standpoint, it should be remembered that compound C is the only candidate to elicit these FOXO/PGC1 effects and to significantly reduce both PSEN and Nicastrin levels (fig. 11). Furthermore, it is this compound that shows a favorable response in reducing the level of phospho-Tau and the level of GSK3b in IMR-32 cells (fig. 12).

Thus, compound C appears to be the compound of choice for eliciting the following triple beneficial effects in neuronal cells: 1) by stimulating overall mitochondrial potential, increasing PGC1 levels, and decreasing FOXO phosphorylation-thereby allowing nuclear entry thereof, resulting in beneficial effects on mitochondrial activity and function; 2) a cryptic role in reducing amyloid accumulation by reducing the expression of PSEN1 and Nicastrin; and 3) reducing the implicit effect of Tau entanglement formation by reducing the level of phosphorylated Tau and the enzyme thought to be responsible for Tau phosphorylation (GSK3 b).

Again, to test the cellular specificity of these effects, protein extracts from CDE combination-treated hepatic AML-12 cells were probed with the same antibodies to detect differences in FOXO phosphorylation and PGC1a levels. As shown in fig. 15 and 16, and discussed in more detail in the later section, the effects seen with these three corresponding compounds confirm that IMR-32 cellular effects are not observed in the liver. In fact, it is believed that the direct opposite effects were observed, i.e. increased FOXO phosphorylation (nuclear export), unchanged levels of PGC1a and unchanged levels of GSK3 b. However, it is clear that these are the types of actions that are expected to occur in the liver, particularly in the case of diabetic subjects, where hepatic glucose output needs to be controlled. Most importantly, hepatic AML-12 cells were found to be non-responsive to treatment with the individual compounds, but, instead, to respond only to the CDE cocktail, completely different from neuronal cells. Again, with respect to the mode of action of these compounds, this points to very well-defined cell specificity and unpredictable cellular responses.

Results and discussion: liver cell

Hepatocyte-specific and PDK 1/AKT-independent upregulation of FOXO phosphorylation by compound CDE combinations and not by compounds alone or their sulfur analogs

As in the case of IMR-32 neuronal cells, the observation that selenium compounds, in this case CDE combinations, increase MT activity and regulate expression of key genes controlled in cellular energy (UCP2) suggests that we investigated the possible effects that this combination of selenium compounds may have on other key genes involved in liver energy metabolism, insulin signaling and cell proliferation. Therefore, Western blot analysis was performed on CDE-treated AML-12 cells.

As previously mentioned, FOXO is the major signaling molecule for gluconeogenesis and insulin sensitivity in the liver. It has been discussed that the function of FOXO proteins is governed by their phosphorylation state. FOXO phosphorylation excludes them from the nucleus, thereby substantially inactivating them. Dephosphorylation allows FOXO proteins to enter the nucleus, so that they can participate in the transcriptional regulation of several key genes involved in energy metabolism. As shown in fig. 15A, it was found that phosphorylated forms of FOXO3 and 4 (pFOXO3 and pFOXO4 protein levels) were significantly elevated after 6hr of CDE rather than HIJ treatment in AML-12 cells. Quantitative analysis indicated that pFOXO3 increased by about 2.5-fold and pFOXO4 increased by about 3.2-fold in CDE-treated AML-12 cells (FIGS. 15B-C). Since FOXO phosphorylation leads to nuclear exclusion to inactivate FOXO in the nucleus, our results suggest that CDE will act as a FOXO inactivator in hepatocytes. Since increased FOXO phosphorylation was not observed in CDE-treated IMR-32 neuronal cells in assays performed simultaneously and under the same experimental conditions (see fig. 13 above), we can conclude that inactivation of FOXO by CDE is hepatocyte-specific.

In summary, our results indicate that the CDE combination will act as a novel hepatocyte-specific FOXO inactivator. Indeed, in IMR-32 neuronal cells, significant and highly significant activation (dephosphorylation) of FOXO protein occurs in response to either single selenium compounds C, D and E or a combination thereof. Therefore, we were able to selectively in neuronal cells in the case of these specific compoundsActivation ofFOXO proteins and their use in hepatocytesDeactivation of the enzyme. The overall importance and novelty of this finding becomes apparent when considering it in the context of glucose processing in type 2 diabetes and alzheimer's disease.

As a simple background, FOXO is located in the nucleus in its activated or non-phosphorylated state, where it binds to the promoter region of glucose 6-phosphatase, and together with other factors such as PGC-1a, increases transcription of glucose-6-phosphatase, thereby increasing the rate of glucose production. Glucose 6-phosphatase catalyzes the last step in gluconeogenesis and glycogenolysis, resulting in the release of glucose from the liver. Therefore, this is crucial in controlling glucose homeostasis, especially in diabetic subjects. Normally, the process of FOXO phosphorylation is directly controlled by another kinase called AKT (protein kinase B). AKT phosphorylates FOXO and drives it away from the nucleus, thereby reducing glucose production by reducing the transcription rate of glucose 6-phosphatase. AKT itself is under downstream control through a small molecule cascade, which begins with insulin binding to its receptor on the cell surface. This initiates a series of events including two other kinases, phosphatidylinositol 3-kinase (PI3K) and phosphoinositide-dependent protein kinase 1 (PDK 1). The entire pathway is termed the insulin/PI 3K/PDK1/Akt pathway, which has the effect of controlling glucose homeostasis through insulin signaling. Under FOXO control, PDK1 phosphorylates and activates Akt, which in turn phosphorylates and inactivates FOXO.

Since PI3K/PDK1/AKT is the major signal transduction pathway upstream of FOXO for insulin-mediated control of glucose production in the liver, we questioned whether the increased FOXO phosphorylation by CDE was due to activation of the PI3K/PDK/AKT signal transduction pathway in hepatocytes. To test this, we examined the protein levels of pdk1, pAKT T308 and S473 and total AKT in CDE-treated hepatocytes. Surprisingly, CDE did not affect PDK1, phosphorylation of AKT or total AKT levels (fig. 15A). We also examined the levels of two other downstream signaling molecules pGSk3a and pGSk3b, which were directly under AKT control, and no change in their protein levels was observed (fig. 15A), consistent with no change in pAKT in CDE-treated cells. The levels of p4Ebp (the downstream molecular target for AKT/mTor signaling) and pElf2be S539 (the downstream molecular target for Gsk 3), which are critical for insulin-driven protein synthesis or translation, were also unaffected (fig. 15A). This provides additional direct molecular evidence that CDE has no toxic effect in affecting hepatocyte proliferation/survival, as previously described.

As described in the case of neuronal IMR-32 cells, PGC1a is an important gene for MT biogenesis and carbohydrate metabolism. In hepatocytes it also acts in concert with FOXO to drive transcription of genes involved in gluconeogenesis, but not in the absence of FOXO in the nucleus. By quantitative analysis after CDE combination treatment, we examined PGC1a protein expression and no significant changes in PGC protein levels were observed in hepatocytes (fig. 15A and D). However, due to the robust effect observed on FOXO phosphorylation in response to CDE, it is almost certain that it will be excluded from the nucleus, and therefore the level of PGC1a becomes less important because FOXO is required to initiate the gluconeogenesis process. Taken together, these results indicate that CDE does not affect PI3k/PDK1/AKT signaling and several other AKT direct or indirect downstream signaling molecules, in addition to the key FOXO described above. In other words, CDE selectively inactivates FOXO in hepatocytes and this effect appears to be independent of PI3K/PDK1/AKT signaling.

Thus, in essence, the mode of action of CDE in hepatocytes may be completely independent of the insulin-driven PI3k/PDK1/AKT signaling pathway, i.e., it may be insulin-independent. Its importance becomes directly apparent to those skilled in the art of metabolic signaling pathways, as it reduces the physiological consequences of hepatocytes becoming insulin-resistant in the context of controlling hepatic glucose output. Bypassing insulin signal transduction while still being able to control glucose homeostasis through FOXO modulation, opens up many therapeutic possibilities for treating diabetes in general; making it less dependent on the administration of exogenous insulin.

Furthermore, the importance of finding potential AKT-independent FOXO inhibitors in the liver should not be underestimated from a broader health perspective. It is well established that the PI3k/PDK1/AKT pathway is the prototype pathway to promote cell growth and is constitutively active in many cancers. AKT, when activated, is inactivated by FOXO and plays a key role in different cellular processes-not only glucose homeostasis. Cancer progression is predominant in these other pathways. In this regard, it is interesting to note that AKT was initially identified as an oncogene in the transformed retrovirus AKT 8. Thus, any compound that can exert a key role for AKT, but does so in an AKT-independent manner, is highly novel and valuable.

Again, to complete our study of compound specificity, we wanted to determine if a single compound in CDE has the ability to inactivate FOXO in hepatocytes, AML-12 cells were treated with the individual compound under the same experimental conditions. Western blot analysis showed that no inactivation of FOXO (indicated by an increase in pFOXO) was observed after 6 or 24 hr treatment in compound-treated hepatocytes alone (fig. 16). We also examined a number of other signaling molecules and found no increase in phosphorylation of AKT, Gsk3a/b and Elf2be in hepatocytes after 6 or 24 hr treatment with C, D or E (fig. 16). The only effect observed was related to compound E, which was shown to slightly reduce the levels of pAKT and pElf2b epsilon S539 after 6 hr treatment in AML-12 cells (fig. 16). However, the effect of compound E on AKT must be unimportant, as this is not reflected by a decrease in compound E-mediated FOXO (direct downstream target of AKT) phosphorylation. Thus, this did not indicate a significant change in potential gluconeogenesis in E-treated cells. Dephosphorylation of EIF2b epsilon may indicate an increased mRNA/protein translation level, but this has not been fully studied. However, it is clear that the protein expression of the species tested in these western blots was unchanged by any of the three selenium compounds (fig. 16). In any event, our results indicate that selenium compound C, D or E alone does not increase FOXO phosphorylation in hepatocytes. Thus, there is a synergistic effect between C, D and the E compound to inactivate FOXO in hepatocytes.

In summary, our results indicate that CDE, but not the individual compounds, will act as hepatocyte-specific and PI 3K/PDK/AKT-independent FOXO inactivators.

Downregulation of G6pc (a glucose-producing FOXO downstream target) expression in AML-12 cells following treatment with the compound CDE selenium compound

As mentioned above, the glucose 6-phosphatase catalytic subunit (G6PC) is a direct downstream target for FOXO, which enhances glucose production, particularly in the liver. Increased pFOXO (inactivation) levels in CDE-treated hepatocytes indicate that CDE may function to control glucose production and increase insulin sensitivity in hepatocytes. True evidence that selenium compounds in combination with CDE can modulate gluconeogenesis or glycogenolysis by FOXO phosphorylation is that reduced expression of G6PC is observed in the liver environment. To test this hypothesis, we examined G6pc expression in hepatocytes by quantitative RT-PCR analysis.

As shown in figure 17, treatment of AML-12 hepatocytes with CDE combinations, but not HIJ sulfur analog combinations, resulted in a very significant 45% reduction in G6pc expression in hepatocytes. Furthermore, we also examined G6pc expression in AML-12 cells after treatment with all compounds alone and did not observe any significant changes (increase or decrease) in G6pc expression (fig. 17); this finding is consistent with the unaltered FOXO phosphorylation levels observed in compound C-, D-or E-treated hepatocytes, as determined by western blot analysis (fig. 16). A reduction in G6pc expression in response to CDE combination was observed in three replicate experiments using different batches of cells (data not shown).

In summary, our results demonstrate that CDE, but not the compound alone, can significantly attenuate G6pc expression, thus representing a new way to reduce hepatic glucose output. Our general findings further point to the mode of action mediated by AKT-independent increase in FOXO phosphorylation of these compounds. These data provide strong in vitro evidence that CDE has significant potential in the therapeutic ability to control hepatic glucose output in obese and type 2 diabetic subjects.

General overview and discussion

We have determined that compound C, but not D, increases MT potential in kidney cells, and that both compounds C and D increase MT potential in mouse skeletal muscle myoblasts C2C12 cells. These results indicate that compound C can be used against progressive renal failure, and that C and D can be used against sarcopenia (sarcopenia) resulting from the progressive loss of MT function in the kidney or skeletal muscle. In the case of skeletal muscle, we also expect C and D to be potentially useful in the T2DM research and control field, given that skeletal muscle utilizes 75-80% of daily glucose uptake and that mitochondrial dysfunction in skeletal muscle is considered a key initiator of T2 DM.

Furthermore, our experiments with human neuronal IMR-32 cells showed that one of these compounds (especially compound C) is uniquely able to combat AD pathogenesis. This conclusion is based on the following important findings:

(1) C briefly raising the MT potential (FIG. 3)

(2) Compound C increased neuronal cell survival (FIG. 5)

(3) Neuronal cell-specific down-regulation of FOXO phosphorylation (important for cellular metabolism) and up-regulation of PGC1a (important for MT biogenesis) (fig. 14 and the hepatocyte data of fig. 16);

(4) for APP cleavage, the γ -secretase complex genes PSEN and Nicastrin were selectively targeted to inhibit their expression (fig. 11);

(5) inhibition of Tau phosphorylation likely antagonizes tangle formation in AD (figure 12); and

(6) neuronal cell-specific GSK3b down-regulation, which again suggests a decreased probability of Tau phosphorylation and decreased tangle formation in the case of AD (fig. 12 and the hepatocyte data of fig. 16).

We have identified a novel combination of hepatocyte-specific and PI3K/PDK 1/AKT-independent FOXO-inactivating Compounds (CDEs) (i.e. a combination of CDEs, not the individual compounds). This combination of compounds significantly down-regulated G6pc expression in hepatocytes by nuclear exclusion of FOXO protein. We expect this combination to be particularly useful in studying and ameliorating the symptoms and pathologies arising from metabolic syndrome, obesity and T2 DM. Our reason is based on the following findings:

(1) significantly increased MT potential in hepatocytes by CDE combination alone, but not any other compounds or their combinations (fig. 7);

(2) Has no toxic effect on survival of liver cells or neuron cells (FIGS. 5 and 9)

(3) Specific and significant down-regulation of the connexin 2 (Ucp2) gene by CDE (fig. 8); of particular note in this regard is the finding from the latest studies that inhibition of UCP2 expression reverses diet-induced diabetes by affecting both insulin secretion and action.

(4) Inactivation (phosphorylation) of hepatocyte-specific FOXO by CDE (neuronal cell data of fig. 15 and 13);

(5) no effect on FOXO inactivation by the compound alone was noted (FIG. 16)

(6) No effect on phosphorylation of PI3K/PDK/Akt signaling by CDE was noted (FIG. 15). This indicates that CDE represents a novel AKT-independent FOXO inactivator.

(7) The key FOXO target gene G6pc was significantly down-regulated by CDE (fig. 17) rather than by any individual compound (fig. 17). The control of hepatic glucose output by FOXO-mediated transcriptional activation of G6pc was previously discussed.

Based on the above experimental data, we believe that the CDE combination can be used specifically as an effective FOXO inactivator in hepatocytes to reduce G6pc expression and reduce hepatic glucose output. Furthermore, we demonstrate that the effect is independent of PI3K/PDK/Akt signaling. A description of how this process may function is shown in fig. 18. Although, normally, FOXO phosphorylation and its subsequent entry into or exclusion from the nucleus is controlled by the insulin/PI 3K/PDK1/AKT pathway, we demonstrate that a specific combination of compounds C, D and E leads to FOXO phosphorylation by yet unidentified kinases. Phosphorylated FOXO excluded from the nucleus failed to transcriptionally activate G6PC, which in turn resulted in decreased glucose output from the liver. A decrease in hepatic glucose output and associated decrease in blood glucose concentration will result in an insulin-sparing effect, i.e. a decreased need for insulin secretion by the pancreas in response to hyperglycemia.

It is also very reasonable to expect that lower levels of blood glucose can lead to both increased insulin sensitivity in peripheral tissues and more controlled insulin release through pancreatic beta-cells. That is, the insulin output through the pancreas will be less likely to be overloaded due to the generally lower circulating glucose levels. From the experiments performed and provided herein, we were unable to identify any negative effects on growth or pathway activation (signally capable of initiating uncontrolled proliferation and growth, i.e., leading to cancer) of all cell types tested. Indeed, the ability of these compounds to bypass the AKT-signaling pathway (at least in the liver) makes them of particular interest from the standpoint of reduced toxicity potential.

Based on the findings provided above, we also believe that we can assume a reliable model for the effect of compound C in particular on improving AD pathology (amyloid plaques and Tau tangles). As shown in fig. 19A-C, key AD-associated genes whose expression is reduced or altered in IMR-32 cells have binding motifs (sites) for FOXO 1, 3 or 4 in their gene promoter regions. This is true for Gsk3B (fig. 19A), Psen1 (fig. 19B), and Nicastrin (fig. 19C). This means that nuclear localized FOXO proteins can bind to these gene promoters and negatively regulate their transcription. Recognizing this point, it is therefore very easy to imagine the situation (fig. 19D) where compound C dephosphorylates FOXO proteins and allows them to enter the nucleus. Here, the active FOXO protein binds to the promoter regions of the above genes and inhibits their transcription. Lower levels of GSK3b (figure 12) in IMR cells would result in decreased Tau phosphorylation, followed as a direct consequence by decreased microtubule stability and decreased tangle formation.

Likewise, FOXO protein binding to the promoter regions of PSEN and Nicastrin inhibits transcription from these genes, which results in lower amounts of these key γ -secretase components being produced in neuronal cells. This naturally implies lower levels of abnormal APP processing, lower amyloid- β peptide concentrations and reduced amyloid plaque burden as a result.

All publications and patents mentioned in this application are herein incorporated by reference. Various modifications and variations of the methods and compositions of the present application will be apparent to those skilled in the art without departing from the scope and spirit of the application. While the present application has been described in connection with certain preferred embodiments, it should be understood that the application as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the application which are obvious to those skilled in the relevant fields are intended to be within the scope of the following claims.

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