MODULATING EXPRESSION LEVEL OF A GENE ENCODING A CYTOCHROME P450 PROTEIN BY TREATING A HUMAN SUBJECT WITH A NITROXIDE

Abstract

A method of treatment is disclosed. The method comprises administering to a human subject having or at risk of developing a disease associated with decreased expression of one or more genes encoding cytochrome P450, an effective amount of a nitroxide antioxidant, wherein the nitroxide antioxidant increases an expression level of one or more genes encoding cytochrome p450 enzymes.

Claims

1. A method of increasing expression level of a gene encoding one or more cytochrome p450 proteins in a subject having a condition selected from the group consisting of non-alcoholic fatty liver disease, hepatocellular carcinoma, hepatic disease, a neurodegenerative disease, cirrhosis, hepatocellular carcinoma and a family history of Alzheimer's Disease, the method comprising: identifying a subject having a decreased expression level of the gene encoding one or more cytochrome p450 proteins associated with a condition selected from the group consisting of non-alcoholic fatty liver disease, hepatocellular carcinoma, hepatic disease, a neurodegenerative disease, cirrhosis, hepatocellular carcinoma and a family history of Alzheimer's Disease; and administering an effective amount of a nitroxide antioxidant to the subject, wherein the nitroxide antioxidant has the general formula ##STR00006## wherein x is wherein X is selected from O— and OH and R is selected from H, OH, and NH.sub.2, wherein the administration of the nitroxide antioxidant increases expression level of the gene encoding one or more cytochrome p450 proteins, and wherein the increased expression level of the gene treats the condition.

2. The method of claim 1, wherein the subject has hepatocellular carcinoma.

3. (canceled)

4. (canceled)

5. The method of claim 1, wherein the subject has a hepatic disease.

6. The method of claim 1, wherein increasing the expression level prevents a neurodegenerative disease.

7. The method of claim 1, wherein the subject has cirrhosis.

8. The method of claim 1, wherein the individual has a family history of Alzheimer's Disease.

9. The method of claim 1, wherein the nitroxide antioxidant is administered before one or more chemotherapeutic agents.

10. The method of claim 1, wherein the subject has a neurodegenerative disease caused by excess brain cholesterol.

11. The method of claim 1, wherein the subject has non-alcoholic fatty liver disease.

12. The method of claim 1, wherein the nitroxide antioxidant is 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL).

13. A method of increasing expression level of a gene encoding one or more cytochrome p450 proteins, the method comprising: administering an effective amount of a nitroxide antioxidant to a subject having or at risk of developing a disease associated with decreased cytochrome p450 expression, wherein the disease is selected from the group consisting of non-alcoholic fatty liver disease, hepatocellular carcinoma, hepatic disease, a neurodegenerative disease, cirrhosis, hepatocellular carcinoma and a family history of Alzheimer's Disease, wherein the gene encoding one or more cytochrome p450 proteins is selected from the group consisting of Cyp2c29, Cyp3a25, Cyp3a11, Cyp2j5, Cyp2c50, Cyp2c55, Cyp2d9, Cyp2e1, Cyp2b9, Cyp3a13, Cyp4f15, Cyp2c70, Cyp46a1, Cyp27a1, and Cyp4v3, and wherein the nitroxide antioxidant increases an expression level of the gene encoding one or more cytochrome p450 proteins.

14. The method of claim 13, wherein the nitroxide antioxidant is 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL).

15. The method of claim 13, wherein the nitroxide antioxidant passes through the blood brain barrier.

16. The method of claim 13, wherein the disease is caused by increased cholesterol.

17. The method of claim 13, wherein the subject has hepatitis.

18. The method of claim 13, wherein the subject is in need of increased xenobiotic metabolism.

19. (canceled)

20. (Canceled)

21. The method of claim 13, wherein the nitroxide antioxidant is selected from the group consisting of 2-ethyl-2,5,5-trimethyl-3-oxazolidine-1-oxyl (OXANO), 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL), 4-amino-2,2,6,6-tetramethyl-1-piperidinyloxy (Tempamine), 3-Amin omethyl-PROXYL, 3-Cyano-PROXYL, 3-Carbamoyl-PROXYL, 3-Carboxy-PROXYL, 4-Oxo-TEMPO, 2,2,6,6-tetramethyl-4-oxo-1-piperidinyloxy (TEMPONE), 1-Hydroxy-2,2,6,6-tetramethyl-4-oxo-piperidine HCl (TEMPONE-H), 1,2-dipalmitoyl-sn-glycero-3-phospho(tempo)choline (TEMPO PC), and (4-[N,N-dimethyl-N-(2-hydroxyethyl)]ammonium-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO Choline).

22. The method of claim 13, wherein the disease is non-alcoholic fatty liver disease

23. The method of claim 1, wherein the gene encoding one or more cytochrome p450 proteins is selected from the group consisting of Cyp2c29, Cyp3a25, Cyp3a11, Cyp2j5, Cyp2c50, Cyp2c55, Cyp2d9, Cyp2e1, Cyp2b9, Cyp3a13, Cyp4f15, Cyp2c70, Cyp46a1, Cyp27a1, and Cyp4v3.

24. (Withdrawn— currently amended) The method of claim 1, wherein the nitroxide antioxidant is selected from the group consisting of 2-ethyl-2,5,5-trimethyl-3-oxazolidine-1-oxyl (OXANO), 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), 4-amino-2,2,6,6-tetramethyl-1-piperidinyloxy (Tempamine), 3-Amin omethyl-PROXYL, 3-Cyano-PROXYL, 3-Carbamoyl-PROXYL, 3-Carboxy-PROXYL, 4-Oxo-TEMPO. 2,2,6, 6-tetramethyl-4-oxo-1-piperidinyloxy (TEMPONE), 1-Hydroxy-2,2,6,6-tetramethyl-4-oxo-piperidine HCl (TEMPONE-H), 1,2-dipalmitoyl-sn-glycero-3-phospho(tempo)choline (TEMPO PC), and (4-[N,N- dimethyl-N-(2-hydroxyethyl)]ammonium-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO Choline).

Description

EXAMPLES

[0132] The following examples are offered to illustrate but not to limit the invention.

[0133] In order to facilitate understanding, the specific embodiments are provided to help interpret the technical proposal, that is, these embodiments are only for illustrative purposes, but not in any way to limit the scope of the invention. Unless otherwise specified, embodiments do not indicate the specific conditions, are in accordance with the conventional conditions or the manufacturer's recommended conditions.

Example 1. Effects of Tempol on Expression of Genes Associated With the Cytochrome p450 Family

[0134] To assess the effects of Tempol on gene expression, Tempol was administered to experimental mice at a dose of 5 mg/g of food from 14 months to 31 months after birth. Mice receiving the same food without the addition of Tempol were used as a negative control. At the age of 31 months, the experimental animals were sacrificed and the hearts were surgically removed. The expression of a broad spectrum of genes in the cardiac tissue was assessed using chip-based microarray technology. Such chips are well known in the art and are widely used to assess gene expression. The experimental results showed that fifteen genes associated with the cytochrome p450 family, Cyp2c29, Cyp3a25, Cyp3a11, Cyp2j5, Cyp2c50, Cyp2c55, Cyp2d9, Cyp2e1, Cyp2b9, Cyp3a13, Cyp4f15, Cyp2c70, Cyp46a1, Cyp27a1, Cyp4v3, and homologues thereof, exhibited statistically significant increase in expression. This result is shown in Table 1.

TABLE-US-00001 TABLE 1 Genes Associated With The cytochrome p450 family Exhibiting Increased Expression In White Adipose Tissue After Tempol Administration Tempol- Control treated Fold Symbol Gene title mice mice change P-value Cyp2c29 Cytochrome P450, 90 1210 13.45 0.01 family 2, subfamily c, polypeptide 29 Cyp3a25 Cytochrome P450, 65 564 8.61 0.02 family 3, subfamily a, polypeptide 25 Cyp3a11 Cytochrome P450, 177 1375 7.77 0.02 family 3, subfamily a, polypeptide 11 Cyp2j5 Cytochrome P450, 26 138 5.37 0.04 family 2, subfamily j, polypeptide 5 Cyp2c50 Cytochrome P450, 41 147 3.58 0.02 family 2, subfamily c, polypeptide 50 Cyp2c55 Cytochrome P450, 15 44 2.97 0.04 family 2, subfamily c, polypeptide 55 Cyp2d9 Cytochrome P450, 72 200 2.78 0.04 family 2, subfamily d, polypeptide 9 Cyp2e1 Cytochrome P450, 2329 5079 2.18 0.02 family 2, subfamily e, polypeptide 1 Cyp2b9 Cytochrome P450, 54 103 1.91 0.03 family 2, subfamily b, polypeptide 9 Cyp3a13 Cytochrome P450, 50 92 1.85 0.04 family 3, subfamily a, polypeptide 13 Cyp4f15 Cytochrome P450, 72 122 1.70 0.03 family 4, subfamily f, polypeptide 15 Cyp2c70 Cytochrome P450, 180 292 1.62 0.03 family 2, subfamily c, polypeptide 70 Cyp46a1 Cytochrome P450, 115 166 1.45 0.04 family 46, subfamily a, polypeptide 1 Cyp27a1 Cytochrome P450, 864 1135 1.31 0.02 family 27, subfamily a, polypeptide 1 Cyp4v3 Cytochrome P450, 739 901 1.22 0.04 family 4, subfamily v, polypeptide 3

Example 2. Treating Age-Related Decrease in Gene Expression

[0135] A 70-kilogram human subject over the age of 65 is identified for decreased expression level of Cyp2c29, Cyp3a25, Cyp3a11, Cyp2j5, Cyp2c50, Cyp2c55, Cyp2d9, Cyp2e1, Cyp2b9, Cyp3a13, Cyp4f15, Cyp2c70, Cyp46a1, Cyp27a1, or Cyp4v3. The human subject is administered a dose of 2000 mg of Tempol per day for 180 days. This may be administered in a single dose, or may be administered as a number of smaller doses over a 24-hour period: for example, three 500-mg doses at eight-hour intervals. Following treatment, the serum level of Cyp2c29, Cyp3a25, Cyp3a11, Cyp2j5, Cyp2c50, Cyp2c55, Cyp2d9, Cyp2e1, Cyp2b9, Cyp3a13, Cyp4f15, Cyp2c70, Cyp46a1, Cyp27a1, or Cyp4v3, is increased.

Example 3. Treating a Human Subject with Decreased Gene Expression

[0136] A 70-kilogram human subject is identified for decreased expression level of Cyp2c29, Cyp3a25, Cyp3a11, Cyp2j5, Cyp2c50, Cyp2c55, Cyp2d9, Cyp2e1, Cyp2b9, Cyp3a13, Cyp4f15, Cyp2c70, Cyp46a1, Cyp27a1, or Cyp4v3. The human subject is administered a dose of 2000 mg of Tempol per day for 180 days. This may be administered in a single dose, or may be administered as a number of smaller doses over a 24-hour period: for example, three 500-mg doses at eight-hour intervals. Following treatment, the serum level of Cyp2c29, Cyp3a25, Cyp3a11, Cyp2j5, Cyp2c50, Cyp2c55, Cyp2d9, Cyp2e1, Cyp2b9, Cyp3a13, Cyp4f15, Cyp2c70, Cyp46a1, Cyp27a1, or Cyp4v3, is increased.

Example 4. Treating a Human Subject With Age-Related Disease

[0137] A 70-kilogram human subject over the age of 65 and having a cardiovascular disease is identified for decreased expression level of Cyp2c29, Cyp3a25, Cyp3a11, Cyp2j5, Cyp2c50, Cyp2c55, Cyp2d9, Cyp2e1, Cyp2b9, Cyp3a13, Cyp4f15, Cyp2c70, Cyp46a1, Cyp27a1, or Cyp4v3. The human subject is administered a dose of 2000 mg of Tempol per day for 180 days. This may be administered in a single dose, or may be administered as a number of smaller doses over a 24-hour period: for example, three 500-mg doses at eight-hour intervals. Following treatment, the serum level of Cyp2c29, Cyp3a25, Cyp3a1 1, Cyp2j5, Cyp2c50, Cyp2c55, Cyp2d9, Cyp2e1, Cyp2b9, Cyp3a13, Cyp4f15, Cyp2c70, Cyp46a1, Cyp27a1, or Cyp4v3, is increased.

Example 5. Treating a Human Subject at Risk of Developing Cancer

[0138] A 70-kilogram human subject at risk of developing colorectal cancer is identified for decreased expression level of Cyp2c29, Cyp3a25, Cyp3a11, Cyp2j5, Cyp2c50, Cyp2c55, Cyp2d9, Cyp2e1, Cyp2b9, Cyp3a13, Cyp4f15, Cyp2c70, Cyp46a1, Cyp27a1, or Cyp4v3. The human subject is administered a dose of 2000 mg of Tempol per day for 180 days. This may be administered in a single dose, or may be administered as a number of smaller doses over a 24-hour period: for example, three 500-mg doses at eight-hour intervals. Following treatment, the serum level of Cyp2c29, Cyp3a25, Cyp3a11, Cyp2j5, Cyp2c50, Cyp2c55, Cyp2d9, Cyp2e1, Cyp2b9, Cyp3a13, Cyp4f15, Cyp2c70, Cyp46a1, Cyp27a1, or Cyp4v3, is increased.

Example 6. Treating a Human Subject at Risk of Developing an Autoimmune Disease

[0139] A 70-kilogram human subject at risk of developing rheumatoid arthritis is identified for decreased expression level of Cyp2c29, Cyp3a25, Cyp3a11, Cyp2j5, Cyp2c50, Cyp2c55, Cyp2d9, Cyp2e1, Cyp2b9, Cyp3a13, Cyp4f15, Cyp2c70, Cyp46a1, Cyp27a1, or Cyp4v3. The human subject is administered a dose of 2000 mg of Tempol per day for 180 days. This may be administered in a single dose, or may be administered as a number of smaller doses over a 24-hour period: for example, three 500-mg doses at eight-hour intervals. Following treatment, the serum level of Cyp2c29, Cyp3a25, Cyp3a11, Cyp2j5, Cyp2c50, Cyp2c55, Cyp2d9, Cyp2e1, Cyp2b9, Cyp3a13, Cyp4f15, Cyp2c70, Cyp46a1, Cyp27a1, or Cyp4v3, is increased.

Example 7. Treating a Human Subject at Risk of Developing a Condition Due to Aging

[0140] A 70-kilogram human subject of 45 years old at risk of developing a condition due to aging is identified. The human subject is administered a dose of 2000 mg of Tempol per day for 180 days. This may be administered in a single dose, or may be administered as a number of smaller doses over a 24-hour period: for example, three 500-mg doses at eight-hour intervals. Following treatment, the serum level of Cyp2c29, Cyp3a25, Cyp3a11, Cyp2j5, Cyp2c50, Cyp2c55, Cyp2d9, Cyp2e1, Cyp2b9, Cyp3a13, Cyp4f15, Cyp2c70, Cyp46a1, Cyp27a1, or Cyp4v3, is increased.

[0141] In at least some of the previously described embodiments, one or more elements used in an embodiment can interchangeably be used in another embodiment unless such a replacement is not technically feasible. It will be appreciated by those skilled in the art that various other omissions, additions and modifications may be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter, as defined by the appended claims.

[0142] With respect to the use of substantially any plural and/or singular terms herein, those having skill in the art can translate from the plural to the singular and/or from the singular to the plural as is appropriate to the context and/or application. The various singular/plural permutations may be expressly set forth herein for sake of clarity.

[0143] It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (e.g., bodies of the appended claims) are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases “at least one” and “one or more” to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles “a” or “an” limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases “one or more” or “at least one” and indefinite articles such as “a” or “an” (e.g., “a” and/or “an” should be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the bare recitation of “two recitations,” without other modifiers, means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to “at least one of A, B, and C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “a system having at least one of A, B, and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). In those instances where a convention analogous to “at least one of A, B, or C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “a system having at least one of A, B, or C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and/or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B.”

[0144] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0145] As will be understood by one skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 articles refers to groups having 1, 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

[0146] While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the following claims.