# US 2014/0193828 A1 · Molecular flux rates through critical pathways measured by stable isotope labeling in vivo, as biomarkers of drug action and disease activity

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- Number: US 2014/0193828 A1
- Document ID: US20140193828A1
- Status: Published application
- Kind: A1
- Application number: 14/201,761
- Filed: 2014-03-07
- Published: 2014-07-10
- Earliest claimed: 2004-02-20
- Assignees: The Regents of the University of California
- Applicants: The Regents of the University of California
- Inventors: Marc K. HELLERSTEIN
- Web page: https://patentgrep.com/patent/US20140193828A1

## CPC

- G01N 33/6881: {from skin}
- G01N 33/5008: {for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics}
- G01N 33/5011: {for testing antineoplastic activity}
- G01N 33/5014: {for testing toxicity}
- G01N 33/5038: {involving detection of metabolites per se}
- G01N 33/5088: {of vertebrates}
- G01N 33/58: involving labelled substances (G01N33/53 takes precedence)
- G01N 33/60: involving radioactive labelled substances
- G01N 33/6887: {from muscle, cartilage or connective tissue}
- G01N 33/6893: {related to diseases not provided for elsewhere}
- G01N 33/92: involving lipids, e.g. cholesterol {, lipoproteins, or their receptors (steroid hormones G01N33/743)}
- G01N 2500/00: Screening for compounds of potential therapeutic value
- G01N 2800/042: Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism
- G01N 2800/044: Hyperlipemia or hypolipemia, e.g. dyslipidaemia, obesity
- G01N 2800/323: Arteriosclerosis, Stenosis
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## 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. In one aspect of the invention, stable isotope-labeled substrate molecules are administered to a subject and the label is incorporated into targeted molecules in a manner that reveals molecular flux rates through one or more metabolic pathways of interest. By this method, a comparison between subjects and control subjects reveals the effects of the chemical entity or entities on the biomarkers. This, in turn, allows for the identification of potential therapeutic uses or toxicities of the compound. Combinations of compounds can also be systematically evaluated for complementary, synergistic, or antagonistic actions on the metabolic pathways of interest, using the methods of the present invention as a strategy for identifying and confirming novel therapeutic or toxic combinations of compounds.

## Claims

### Claim at position 1

1-36. (canceled)

### Claim 37

A method for evaluating an action of one or more compounds on a molecular flux rate through a solid or liquid tumor formation 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 DNA molecule derived from the solid or liquid tumor to produce at least one first isotope-labeled DNA molecule derived from the solid or liquid tumor within said solid or liquid tumor formation 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 DNA molecule derived from the solid or liquid tumor;
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 DNA molecule derived from the solid or liquid tumor;
d) calculating a molecular flux rate through said solid or liquid tumor formation 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 DNA molecule derived from the solid or liquid tumor;
e) exposing a second living system to said one or more compounds; 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 DNA molecule derived from the solid or liquid tumor to produce at least one second isotope-labeled DNA molecule derived from the solid or liquid tumor;
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 DNA molecule derived from the solid or liquid tumor;
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 DNA molecule derived from the solid or liquid tumor;
i) calculating a molecular flux rate through said solid or liquid tumor formation 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 DNA molecule derived from the solid or liquid tumor; and
j) comparing said molecular flux rate through said solid or liquid tumor formation pathway in said first living system to said molecular flux rate through said solid or liquid tumor formation pathway in said second living system to evaluate the action of said one or more compounds on said molecular flux rate through the solid or liquid tumor formation pathway in said second living system.

### Claim 38 (depends on 37)

The method of claim 37, wherein the molecular flux rate through said solid or liquid tumor formation pathway is an indicator of tumor growth, grade, prognosis, aggressiveness, or therapeutic treatment thereof.

### Claim 39 (depends on 38)

The method of claim 38, wherein the molecular flux rate through said solid or liquid tumor formation 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 tumor growth, grade, prognosis, aggressiveness, or therapeutic treatment thereof.

### Claim 40 (depends on 38)

The method of claim 38, wherein the molecular flux rate through said solid or liquid tumor formation pathway contributes to the prognosis, survival, morbidity, mortality, stage, therapeutic response, symptomology, disability or other clinical factor of tumor growth, grade, prognosis, aggressiveness, or therapeutic treatment thereof.

### Claim 41 (depends on 38)

The method of claim 38, wherein the molecular flux rate through said solid or liquid tumor formation pathway in said first living system and the molecular flux rate through said solid or liquid tumor formation pathway in said second living system are measured concurrently.

### Claim 42 (depends on 41)

The method of claim 41, wherein said concurrent measurement is achieved by use of stable isotopic labeling techniques.

### Claim 43 (depends on 42)

The method of claim 42, wherein said first isotope-labeled substrate and said second isotope-labeled substrate are stable, non-radioactive isotope-labeled substrates.

### Claim 44 (depends on 43)

The method of claim 43, wherein said first isotope-labeled substrate and said second isotope-labeled substrate are both stable isotope-labeled water.

### Claim 45 (depends on 44)

The method of claim 44, wherein the stable isotope-labeled water is 2H2O.

### Claim 46 (depends on 41)

The method of claim 41, rein said concurrent measurement is achieved by use of radioisotope labeling techniques.

### Claim 47 (depends on 37)

The method of claim 37, 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.

### Claim 48 (depends on 47)

The method of claim 47, wherein said first living system and said second living system are both a human.

### Claim 49 (depends on 37)

The method of claim 37, wherein said first isotope-labeled substrate and said second isotope-labeled substrate are each independently selected from the group consisting of 2H2O, 2H-glucose, 2H-labeled amino acids, 2H-labeled organic molecules, 13C-labeled organic molecules, 13CO2, 15N-labeled organic molecules, 3H2O, 3H-labeled glucose, 3H-labeled amino acids, 3H-labeled organic molecules, 14C-labeled organic molecules, and 14CO2.

### Claim 50 (depends on 37)

The method of claim 37, wherein said isotope labeled substrate is 2H2O.

### Claim 51 (depends on 37)

An isotope-labeled DNA molecule derived from a solid or liquid tumor, wherein the isotope-labeled DNA molecule is generated by the method of claim 37.

### Claim 52 (depends on 37)

The method of claim 37, wherein said first living system and said second living system are different individual living systems of the same species.

### Claim 53 (depends on 37)

The method of claim 37, wherein said first living system is said second living system prior to exposure to said one or more compounds.

### Claim 54 (depends on 53)

The method of claim 53, wherein said first living system and said second living system are both the same individual human subject.

### Claim 55 (depends on 37)

The method of claim 37, wherein the DNA molecule derived from the solid or liquid tumor is from breast, colon, lung, or lymphoma tissue.