S-adenosyl methionine regulation of metabolic pathways and its use in diagnosis and therapy

• US 6,020,139 A
• Filed: 04/25/1995
• Issued: 02/01/2000
• Est. Priority Date: 04/25/1995
• Status: Expired due to Fees

• Current Assignee: Mediquest Therapeutics, Inc.
• Original Assignee: Oridigm Corporation
• Inventors: O'Day, Christine L., Vermeulen, Nicolaas M. J., Schwartz, Dennis E.
• Primary Examiner(s): Housel, James C.
• Assistant Examiner(s): Swartz, Rodney P.
• Application Number:
  US08/428,963
• Time in Patent Office: 1,743 Days
• Field of Search: 435/7.1, 435/192, 514/556
• US Class Current: 506/7
• CPC Class Codes: A61K 31/198 Alpha-amino acids, e.g. alanine or edetic acid [EDTA] betaine A61K31/205; proline A61K31/401; tryptophan A61K31/405; histidine A61K31/4172; peptides not degraded to individual amino acids A61K38/00
• CPC Class Codes: A61K 31/4188 condensed with other heterocyclic ring systems, e.g. biotin, sorbinil
• CPC Class Codes: A61K 31/455 Nicotinic acids, e.g. niacin; Derivatives thereof, e.g. esters, amides
• CPC Class Codes: A61K 31/7076 containing purines, e.g. adenosine, adenylic acid
• CPC Class Codes: A61K 38/1709 from mammals
• CPC Class Codes: A61K 45/06 Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca

Abstract

A new paradigm of disease centers around the metabolic pathways of S-adenosyl-L-methionine (SAM), the intermediates of these pathways and other metabolic pathways influenced by the SAM pathways. Methods are provided to analyze and modulate SAM pathways associated with a disease or condition. Such methods permit identification and utilization of diagnostic and therapeutic protocols and agents for such disease states and conditions.

Claims

1. A method to identify a therapeutic composition or protocol which ameliorates a disease or undesired condition in a subject, which method relies upon recognition of the existence of, and the interconnections between, eight SAM pathways shown in FIGS. 9, herein, which method comprises:

(a) determining the presence of said disease or condition in said subject;

(b) identifying any abnormalities in each of said eight SAM pathways and optionaily in the interconnections therebetween by determining

(i) concentrations of metabolites, and/or

(ii) concentrations or activities of enzymes, and/or

(iii) levels of cellular functions,

that are participants in, or are results of, SAM pathways thereby obtaining a data set of the differences of said concentrations, activities, and/or levels from corresponding normal concentrations, activities, and/or levels,

(c) analyzing said data set by ascertaining, for each difference in concentration, activity, and/or level the position or function of the corresponding metabolite, enzyme or cellular function in said eight SAM pathways or the interconnections therebetween, so as to determine which of one or more metabolites and/or enzymes and/or cellular functions of said SAM pathways is a causative agent of said differences in concentrations, activities or levels in said data set; and

(d) identifying a therapeutic composition or protocol which acts to restore said pathways toward normality, so as to ameliorate said disease or condition,

thereby identifying a composition or protocol that ameliorates said disease or condition.

3. A method according to claim 1 or 2 wherein said disease or condition is selected from the group consisting of a wound, cancer, multiple sclerosis, Alzheimer'"'"'s disease, Parkinson'"'"'s disease, depression, atherosclerosis, cystic fibrosis, diabetes, obesity, Crohn'"'"'s disease and altered circadian rhythmicity.

4. A method according to claim 3, wherein said condition is a wound, said metabolites comprise one or more of SAM, ethylene, biotin, polyamines, nicotinamide, queuine and queuosine.

5. A method according to claim 3, wherein said disease is cancer and said metabolites, enzyme activities or cellular functions comprise one or more of DNA methylation, queuosine and wye-base incorporation into tRNA, SAM synthesis, polyamine synthesis, ethylene synthesis, biotin, methylthioadenosine and 5-deoxyadenosine.

6. A method according to claim 3, wherein said disease is multiple sclerosis and said metabolites or enzyme activities comprise one or more of methyl transferase activity and S-adenosyl homocysteine.

7. A method according to claim 3, wherein said disease is Alzheimer'"'"'s disease and said metabolites, enzyme activities or cellular functions comprise one or more of methylation levels, SAM, biotin, polyamines, folate and vitamin B₂.

8. A method according to claim 3, wherein said disease is Parkinson'"'"'s disease and said metabolites or activities comprise one or more of polyamines, nonspecific N-methylase, acetyl-L-carnitine, Ca²⁺ /calmodulin-dependent protein kinase II, lysolecithin, sphingomyelin, SAM and vitamin B₁₂.

9. A method according to claim 3, wherein said disease is atherosclerosis and said metabolites, enzyme activities or cellular functions comprise one or more of methylation levels, polyamines, acetyl-L-carnitine, calmodulin, and essential phospholipids.

10. A method according to claim 3, wherein said disease is cystic fibrosis said metabolites, enzyme activities or cellular functions comprise one or more of calmodulin, polyunsaturated fatty acids, total saturated and monounsaturated fatty acids, and cystic fibrosis transmembrane conductance regulator.

11. A method according to claim 3, wherein said condition is obesity said metabolites, enzyme activities or cellular functions comprise one or more of methylation levels, serotonin, calmodulin and carnitine.

12. A method according to claim 1, wherein the said data set is obtained by measurements performed on a biological sample from said subject.

13. A method according to claim 1 or 2, wherein said identifying step (b) comprises determining in said biological sample:

(1) the concentration of metabolites selected from the group consisting of;

(A) for Pathway 1, S-adenosylinethionine (SAM), S-adenosyl homocysteine (SAH), adenosine, homocysteine, glutathione, glutathione disulfide, cystathionine, α

-ketobutyrate, cysteine, cystine, taurine, choline, betaine, dimethylglycine, methylglycine, glycine, serine, folate, tetrahydrofolate, methylene tetrahydrofolate, methyltetrahydrofolate, adenosine triphosphate, methylated vitamin B12, an O- or N-methylated product of a methylating enzyme and a demethylateci product of a demethylating enzyme,

(B) for Pathway 2, SAM decarboxylated adenosylmethionine, methylthioadenosine, ornithine, putrescine, spermine, spermidine, N¹ -acetylspermidine, and N¹ -acetylspermine; and

(C) for Pathway 8, biotin, biocytin, and biotin-protein conjugates,

(2) the concentration or activity of enzymes, or the level of cellular functions, selected from the group consisting of;

(A) for Pathway 1, adenosylinethionine synthetase, N⁵ -methyltetrahydrofolate;

homocysteine methyltransferase, betaine;

homocysteine methyltransferase, cystathionine β

-syntdase, γ

-cystathionase, methylene tetrahydrofolate reductase, SAH hydrolase, an O-methyltransferase, an N-methyltransferase, adenosine deaminase, a glutathione synthetic enzyme and a glutathione degradative enzyme;

(B) for Pathway 2, adenosinemethionine decarboxylase, spermidine synthetase, spermine synthetase, spermidine-spermine N¹ -acetyltransferase, polyamine oxidase, methylthioadenosine phosphorylase, ornithine decarboxylase and polyamine transport; and

(C) for Pathway 8, biotinase, pyruvate carboxylase, acetyl-CoA carboxylase, propionyl-CoA carboxylase, 3-methylcrotonyl-CoA carboxylase, geranyl-CoA carboxylase, mixed function oxidase, L-cysteine sulfonate decarboxylase and biotin transport,

thereby obtaining said first data set of differences from normal concentrations of netabolites, concentrations of enzymes, activities of enzymes or cellular functions.

14. A method according to claim 1, 3 or 12 wherein said cellular function is selected from the group consisting of:

(i) binding of a metabolite to receptors therefor, (ii) transport of a metabolite, (iii) membrane fluidity, (iv) membrane potential, (v) cell growth and (vi) programmed cell death.

15. A method according to claim 12, wherein the identifying of step (b) is performed for at least one metabolite or enzyme concentration, enzyme activity or cellular function level by subjecting said biological sample to affinity chromatographic separation using immobilized affinity ligands, which separation comprises the steps of:

(A) contacting the sample with one or more immobilized ligands having binding specificity for said metabolite or said enzyme, and allowing material in the sample to bind to the ligand;

(B) removing any material of the sample not bound to the ligand;

(C) eluting bound material from the ligand to produce an eluate, in a single fraction or as sequential fractions eluted with increasing salt concentrations or with different competitive ligands which compete for binding to said metabolite or enzyme;

(D) identifying the presence and measuring the amount of any bound and eluted material in the eluate;

(E) performing steps (ii)(A)-(D) with a corresponding biological sample from a normal subject not having diabetes; and

(F) comparing the results of step (D) with the results of step (E),

wherein an increase or a decrease in the amount of said metabolite or enzyme in said eluate in step (D) compared to step (E) identifies said abnormal metabolite and/or enzyme and thereby identifies an abnormal SAM pathway or pathways.

16. A method according to claim 12, wherein the identifying of step (b) is performed for at least one metabolite or enzyme concentration, enzyme activity or cellular function level by subjecting said biological sample to a labeling analysis of metabolites comprising the steps of:

(A) contacting said sample with detectably labeled SAM or a detectably labeled first metabolite of SAM, whereby the label from SAM or from said labeled first metabolite is donated to an acceptor molecule in one of said SAM pathways, said acceptor molecule being a second metabolite that is in a SAM pathway distinct from the SAM pathway of said labeled first metabolite;

(B) isolating and measuring the amount of said labeled acceptor molecule produced in step (A), thereby identifying and quantifying said labeled second metabolite;

(C) performing steps (A) and (B) with a sample from a normal subject not having diabetes or with another appropriate control or reference sample; and

(D) comparing the results of step (B) with the results in step (C).

17. A method according to claim 1, 2 or 12 wherein, in step (d), said identifying of said composition is performed by screening combinatorial libraries of candidate compounds.

18. A method according to claim 13, wherein, in step (d), said identifying of said composition is performed by screening combinatorial libraries of candidate compounds.

2. A method to identify a therapeutic composition or protocol which ameliorates a disease or undesired condition in a subject, which method relies upon recognition of the existence of, and the interconnections between, eight SAM pathways shown in FIGS. 2-9, herein, which method comprises:

(a) determining the presence of said disease or condition in said subject,

(b) identifying ay abnormalities in each of said eight SAM pathways and in the interconnections therebetween by determining

(i) concentrations of metabolites, and/or

(ii) concentrations or activities of enzymes, and/or

(iii) levels of cellular functions,

that are participants in, or are results of, SAM pathways thereby obtaining a data set of the differences from normal concentrations, activities, and/or levels from corresponding normal concentrations, activities, and/or levels;

(c) analyzing said data set by ascertaining, for each difference in concentration, activity, and/or level, the position or function of the corresponding metabolite, enzyme or cellular function in said eight SAM pathways or the interconnections therebetween, so as to determine which of one or more metabolites and/or enzymes and/or cellular functions of said SAM pathways is a causative agent of said differences in concentrations, activities or levels in said data set; and

(d) identifying a therapeutic composition or protocol which acts to restore said pathways toward normality, so as to ameliorate said disease or condition,

wherein said data set is converted into digital computer-readable form.