Molecular flux rates through critical pathways measured by stable isotope labeling in vivo, as biomarkers of drug action and disease activity

Patent metadata

Field	Value
Publication number	US-9778268-B2
Application number	US-201414201758-A
Country	US
Kind code	B2
Filing date	Mar 7, 2014
Priority date	Feb 20, 2004
Publication date	Oct 3, 2017
Grant date	Oct 3, 2017

Abstract

The methods described herein enable the evaluation of compounds on subjects to assess their therapeutic efficacy or toxic effects. The target of analysis is the underlying biochemical process or processes (i.e., metabolic process) thought to be involved in disease pathogenesis. Molecular flux rates within the one or more biochemical processes serve as biomarkers and are quantitated and compared with the molecular flux rates (i.e., biomarker) from control subjects (i.e., subjects not exposed to the compounds). Any change in the biomarker in the subject relative to the biomarker in the control subject provides the necessary information to evaluate therapeutic efficacy of an administered drug or a toxic effect and to develop the compound further if desired.

First claim

I claim:

1. A method for evaluating an action of one or more compounds on a molecular flux rate through a skin keratin synthesis pathway in a living system, said method comprising:
a) administering a first isotope-labeled substrate to a first living system, not exposed to said one or more compounds, for a period of time sufficient for said first isotope-labeled substrate to enter into and label at least one skin keratin to produce at least one first isotope-labeled skin keratin within said skin keratin synthesis pathway in said first living system;
b) obtaining one or more samples from said first living system, wherein said one or more samples comprise said at least one first isotope-labeled skin keratin;
c) measuring an isotopic content, rate of incorporation, and/or pattern or rate of change in isotopic content and/or pattern of isotope labeling of said at least one first isotope-labeled skin keratin;
d) calculating a molecular flux rate through said skin keratin synthesis pathway based on the isotopic content, rate of incorporation, and/or pattern or rate of change of isotopic content and/or pattern of isotopic labeling in said at least one first isotope-labeled skin keratin;
e) exposing a second living system to said one or more compounds;
f) administering a second isotope-labeled substrate to said second living system for a period of time sufficient for said second isotope-labeled substrate to enter into and label at least one skin keratin to produce at least one second isotope-labeled skin keratin;
g) obtaining one or more samples from said second living system, wherein said one or more samples comprise said at least one second isotope-labeled skin keratin;
h) measuring an isotopic content, rate of incorporation, and/or pattern or rate of change in isotopic content and/or pattern of isotope labeling of said at least one second isotope-labeled skin keratin;
i) calculating a molecular flux rate through said skin keratin synthesis pathway in said second living system based on the isotopic content, rate of incorporation, and/or pattern or rate of change in isotopic content and/or pattern of isotope labeling of said at least one second isotope-labeled skin keratin; and
j) comparing said molecular flux rate through said skin keratin synthesis pathway in said first living system to said molecular flux rate through said skin keratin synthesis pathway in said second living system to evaluate the action of said one or more compounds on said molecular flux rate through the skin keratin synthesis pathway in said second living system.

2. The method of claim 1 , wherein the molecular flux rate through said skin keratin synthesis pathway is an indicator of psoriasis or skin barrier disease.

3. The method of claim 2 , wherein the molecular flux rate through said skin keratin synthesis pathway contributes to the initiation, progression, severity, pathology, aggressiveness, grade, activity, disability, mortality, morbidity, disease sub- classification or other underlying pathogenic or pathologic feature of psoriasis or skin barrier disease.

4. The method of claim 2 , wherein the molecular flux rate through said skin keratin synthesis pathway contributes to the prognosis, survival, morbidity, mortality, stage, therapeutic response, symptomology, disability or other clinical factor of psoriasis or skin barrier disease.

5. The method of claim 2 , wherein the molecular flux rate through said skin keratin synthesis pathway in said first living system and the molecular flux rate through said skin keratin synthesis pathway in said second living system are measured concurrently.

6. The method of claim 5 , wherein said concurrent measurement is achieved by use of stable isotopic labeling techniques.

7. The method of claim 6 , wherein said first isotope-labeled substrate and said second isotope-labeled substrate are stable, non-radioactive isotope-labeled substrates.

8. The method of claim 7 , wherein said first isotope-labeled substrate and said second isotope-labeled substrate are both stable isotope-labeled water.

9. The method of claim 8 , wherein the stable isotope-labeled water is 2 H 2 O.

10. The method of claim 5 , wherein said concurrent measurements achieved by use of radioisotope labeling techniques.

11. The method of claim 1 , wherein said first living system and said second living system are selected from the group consisting of a prokaryotic cell, a eukaryotic cell, a cell line, an isolated tissue preparation, a rabbit, a dog, a mouse, a rat, a guinea pig, a pig, a non-human primate, and a human.

12. The method of claim 11 , wherein said first living system and said second living system are both a human.

13. The method of claim 1 , wherein said first isotope-labeled substrate and said second isotope-labeled substrate are each independently selected from the group consisting of 2 H 2 O, 2 H-glucose, 2 H-labeled amino acids, 2 H-labeled organic molecules, 13 C-labeled organic molecules, 13 CO 2 , 15 N-labeled organic molecules, 3 H 2 O, 3 H-labeled glucose, 3 H-labeled amino acids, 3 H-labeled organic molecules, 14 C-labeled organic molecules, and 14 CO 2 .

14. The method of claim 1 , wherein said isotope labeled substrate is 2 H 2 O.

15. An isotope-labeled skin keratin generated by the method of claim 1 .

16. The method of claim 1 , wherein said first living system and said second living system are different individual living systems of the same species.

17. The method of claim 1 , wherein said first living system s said second living system prior to exposure to said one or more compounds.

18. The method of claim 17 , wherein said first living system and said second living system are both the same individual human subject.

19. The method of claim 1 , wherein the skin keratin comprises type I and/or type II keratin.

Assignees

• Univ California

Inventors

• Hellerstein Marc K

Classifications

• G01N33/5088 Primary

  of vertebrates · CPC title

• G01N33/6881 Primary

  from skin · CPC title

• G01N2800/042

  Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism · CPC title

• G01N33/6893

  related to diseases not provided for elsewhere · CPC title

• G01N2800/044

  Hyperlipemia or hypolipemia, e.g. dyslipidaemia, obesity · CPC title