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WO2003014699A2 - Compositions et methodes pour detecter et quantifier l'activite de la cox-2 par le metabolisme d'acides lipoamines - Google Patents

Compositions et methodes pour detecter et quantifier l'activite de la cox-2 par le metabolisme d'acides lipoamines Download PDF

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WO2003014699A2
WO2003014699A2 PCT/US2002/024864 US0224864W WO03014699A2 WO 2003014699 A2 WO2003014699 A2 WO 2003014699A2 US 0224864 W US0224864 W US 0224864W WO 03014699 A2 WO03014699 A2 WO 03014699A2
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cox
sample
activity
subject
detecting
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WO2003014699A8 (fr
WO2003014699A3 (fr
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Lawrence J. Marnett
Jeffery J. Prusakiewicz
Kevin R. Kozak
Philip J. Kingsley
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Vanderbilt University
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Vanderbilt University
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Priority claimed from US09/924,082 external-priority patent/US20020106707A1/en
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Publication of WO2003014699A8 publication Critical patent/WO2003014699A8/fr
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/88—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving prostaglandins or their receptors

Definitions

  • the present invention relates generally to the cyclooxygenases and their roles in human pathology, including cancer and inflammation. More particularly, this invention pertains to methods and articles of manufacture for detecting or measuring COX-2 activity by detecting and measuring COX-2 specific enzymatic products.
  • COX is a prostaglandin endoperoxide synthase enzyme (cyclooxygenase, COX, EC 1.14.99.1), which catalyzes the conversion of arachidonic acid to prostaglandin (PG) H2.
  • PG prostaglandin
  • Two isoforms of COX are known, COX-1 and COX-2.
  • COX-1 is constitutively expressed.
  • COX-2 is inducible in a variety of cells, especially those of the central nervous and immune systems (Masferrer et al. 1994, Pro Natl. Acad. Sci. USA 91 :3228-3232; Vane et al. 1994, Pro Natl. Acad. Sci. USA 91 :2046-2050; Kennedy et al. 1993, Biochem.
  • COX-2 activity is associated with a variety of human inflammatory diseases. These diseases include, but are not limited to, acute appendicitis, asthma, myocardial infarction, certain immunological disease processes, infection, malignancy, endotoxemia and reperfusion injury.
  • COX-2 inappropriate expression or over-expression is associated with certain types of cancers, including, but not limited to, carcinoma of the colon, rectum, stomach, esophagus, lung, and skin. The amount of COX-2 expression is related to the stage or progression of cancer (Fosslien, E, et al. 2000, Ann. Clin. Lab.
  • COX-2 has become a major pharmaceutical target for developing treatments for these and other diseases.
  • Nonsteroidal anti-inflammatory drugs prevent hyperalgesia and inflammation by inhibiting the cyclooxygenase-2 (COX-2) catalyzed oxygenation of arachidonic acid to prostaglandin (PG) H 2 .
  • COX-2 cyclooxygenase-2
  • the lipoamino acid N- arachidonylglycine (NAGly) has also been shown to suppress tonic inflammatory pain, and is naturally present at significant levels in many of the same mammalian tissues that express COX-2.
  • the present inventors have discovered that COX-2 selectively metabolizes NAGly to PGH 2 glycine (PGH 2 -Gly) and hydroxyeicosatetraenoic glycine (HETE-Gly).
  • NAGly is a charged arachidonyl derivative that is a selective substrate for COX-2, allowing for easier detection in some instances. Accordingly, the present inventors have discovered the role for COX-2 in the regulation of lipoamino acid levels, including NAGly levels, and the formation of a novel class of eicosanoids from NAGly metabolism.
  • U.S. patent 5,475,021 to Marnett et al. describes a method of measuring the activity of purified COX-2 by measuring ⁇ 2-uptake during catalysis. This method requires purification of the enzyme.
  • U.S. Patent 6,045,773 to Isakson et ah describes a method for measuring COX-2 activity in a mammal by administering a positron-emitting radioisotope-labeled COX-2 selective binding agent to the mammal and then detecting the label by positron-emission tomography (PET).
  • PET positron-emission tomography
  • the present invention provides, in part, novel compositions, articles of manufacture including assays and kits, and methods for detecting and measuring COX-2 enzymatic activity.
  • COXs Cyclooxygenases
  • PG prostaglandin
  • AA arachidonic acid
  • PGH 2 the precursor to various PGs and thromboxanes
  • two COX isoforms have been identified that are structurally and mechanistically similar, but differ mainly in their regulation and tissue distribution [2,3].
  • COX-1 is constitutively expressed in most tissues where it is considered to play a role in various physiologic functions.
  • COX-2 is inducible and tightly regulated by a range of stimuli, including cytokines and mitogens [4,5].
  • PG biosynthesis in the central nervous system and inflammatory cells is primarily attributed to COX-2, explaining in part the observed analgesic and anti-inflammatory properties of nonsteroidal anti-inflammatory drugs (NSAIDs) that inhibit COX-2 [2,6-9].
  • NSAIDs nonsteroidal anti-inflammatory drugs
  • AEA and 2-AG are endogenous ligands for the cannabinoid receptors, and exhibit analgesic and anti-
  • the present invention may be used to provide a system for distinguishing between COX-2 activity and COX-1 activity.
  • systems and methods are disclosed to exploit a COX-2 selective enzymatic reaction with lipoamino acids such as NAGly for specifically detecting and measuring COX-2 enzymatic activity.
  • COX-2 enzymatic activity is detected or measured by detecting or measuring novel PGH2 metabolites, including, but not limited to: PGH 2 -EA, PGB2-EA, PGD 2 -EA, PGE2-EA, PGF 2 ⁇ -EA, TxB 2 - EA, 6-keto-PGFi ⁇ -EA, 15-keto-PGE 2 -EA, 13, 14-dihydro-15- keto-PGE -EA, PGG2-EA, PGH2-EA, PGA2-EA, PGJ2-EA, PGJ 2 - EA derivatives, bicyclo-PGE 2 -EA, TxA 2 -EA and PGI 2 -EA.
  • novel PGH2 metabolites including, but not limited to: PGH 2 -EA, PGB2-EA, PGD 2 -EA, PGE2-EA, PGF 2 ⁇ -EA, TxB 2 - EA, 6-keto-PGFi ⁇
  • the COX-2 specific enzymatic activity is compared to COX-1 activity, for example, a COX- 2 /COX-1 ratio may be analyzed.
  • the inventors provide standards for determining a relative or absolute measurement of COX-2 activity by comparison to a standard or a standard curve generated using the standard.
  • a standard can be used to generate a standard curve for normalization of particular test results.
  • a determination of the amount of COX-2 activity, by the measurement of PGH2-EA, or PGH 2 -Gly metabolites, is used to detect inflammation or cancer.
  • NAGly N- arachidonylglycine
  • NAGly N-arachidonyl-alanine
  • ⁇ - arachidonyl aminobutuyic acid induce analgesia in mice and suppresses formalin-induced tonic inflammatory pain in rats [16, 17].
  • NAGly is structurally analogous to AEA, but lacks affinity for the cannabinoid CB1 receptor [18] (Fig. 1).
  • NAGly and its downstream metabolites are naturally present at significant levels in many of the same mammalian tissues that express COX-2 including the brain, spinal cord, and kidney [17].
  • NAGly As the first selective substrate for COX-2 that bears a charge.
  • the products of NAGly oxygenation are PGH 2 - glycine (PGH 2 -Gly) and hydroxyeicosatetraenoic glycine (HETE-Gly), together representing the first members of a novel class of amino acid eicosanoids.
  • Site-directed mutagenesis indicates that Arg-513 is a critical determinant of COX-2-selective oxygenation of NAGly.
  • an embodiment of the present invention is a method of detecting an activity of a COX-2 enzyme in a
  • 11672N:021089:498744:1:NASHVILLE 7 subject that comprises obtaining a sample of the subject; and detecting an amino acid eicosanoid metabolite in the sample.
  • the presence of the amino acid eicosanoid metabolite indicates the activity of the COX-2 enzyme of the subject.
  • Another embodiment of the present invention is a method of detecting an activity of a COX-2 enzyme in a subject that comprises obtaining a first sample of the subject; detecting a first level of an lipid mediator substrate; administering a COX-2 inhibitor; allowing a period of time to pass; obtaining a second sample of the subject detecting a second level of an lipid mediator substrate; and comparing the first level of the lipid mediator substrate with the second level.
  • Another embodiment of the present invention is a method for screening for a tumor or inflammation in a subject in need thereof that comprises obtaining a sample in a subject; measuring the amount of PGH2-Gly or HETE-Gly metabolite in the sample; and relating the amount measured to an existence of a tumor or inflamation in a subject.
  • Yet another embodiment of the present invention is a method of detecting an activity of COX-2 in a subject that comprises obtaining a sample of the subject; and detecting a COX-2 specific metabolite of an lipoamino acid in the sample, wherein the presence of the COX-2 specific metabolite in the sample indicates the activity of COX-2 in the subject.
  • another embodiment of the present invention is a method of detecting an activity of COX-2 in a subject that comprises obtaining a sample of the subject; detecting a level of a lipid mediator substrate.
  • the level of the lipid mediator substrate is indicative of the COX-2 activity.
  • this embodiment may further include at least one of the following steps: administering a COX-2 inhibitor; allowing a period of time to pass; obtaining a second sample of the subject; detecting a second level of a lipid mediator substrate; and comparing the first measured level of the lipid mediator substrate with the second measured level of the lipid mediator substrate to detect the COX-2 activity in the subject.
  • FIG. 1 shows the structure of various COX-2 Substrate Structures.
  • Figure 2 shows oxygenation of NAGly by COX-2.
  • the vertical bar represents 5 ⁇ M O2.
  • Figure 3 shows mass spectrometry of oxygenated NAGly products.
  • Chemical structures indicate possible assignments for the most abundant products with the appropriate mass-to-charge ratio.
  • Figure 4 shows oxygenation of AA and NAGly by constriction site mutants of COX-2.
  • Initial O2 uptake rates by wild-type and mutant mCOX-2 enzymes (200 nM) with AA (100 ⁇ M) and NAGly (200 ⁇ M) are shown, and are normalized to the
  • Figure 5 shows oxygenation of AA and NAGly by side pocket mutants of COX-2.
  • Figure 6 shows a model of NAGly in the COX-2 active site. A stereo view of the predicted interactions between NAGly and the active site residues of mCOX-2.
  • the present invention provides novel compositions, methods and articles of manufacture for detecting and measuring cyclooxygenase-2 (COX-2) activity in a subject or a sample thereof.
  • Certain aspects of the present invention focus on detecting and measuring metabolites of AEA, such as, but not limited to, PGE 2 -EA, PGD2-EA and TXA2-EA, and preferably metabolites of lipoamino acids such as, but not limited to PGH2- glycine and HETE-Gly.
  • the present invention provides novel compositions, methods and kits for detecting and measuring COX-2 activity, methods for identifying tumors in a subject, evaluating relative tumor severity, and following tumor response to therapy, and methods for detecting inflammation in a subject and evaluating relative inflammation severity. No aspect, embodiment or element, including the claims, of the present invention is bound by theory or mechanism.
  • in vitro includes the meaning of processes occurring in systems wholly or partially purified from the natural environment, such as with purified enzymes or defined enzyme systems.
  • Purified means partially or wholly isolated away from the natural milieu of factors normally associated with a particular macromolecular species.
  • the purified factor comprises 50 percent or more (on a molar basis) of all macromolecular species present in the isolated form.
  • a purified composition will comprise more than about 80 percent of all macromolecular species present.
  • a purified composition comprises more than about 90 percent of all macromolecular species present.
  • the object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species. Solvent species, small molecules ( ⁇ 500 Daltons), and elemental ion species are not considered macromolecular species.
  • "purified" is based upon dry weight and the same percent purities stated above are embodied.
  • COX-2 selective substrate is a substrate that is transformed to an enzymatic reaction product by the COX-2 enzyme; but is not transformed, or is not significantly transformed, to a reaction product by the COX-1 enzyme. It is most preferred that a COX-2 selective substrate of the present invention is not enzymatically transformed to a reaction product by COX-1.
  • COX-1 may have some activity on the COX-2 selective substrate, but it is not significant relative to the COX-2 activity. Relatively insignificant activity can be determined, for example, by measuring the ratio of substrate oxygenation using purified COX-1 and COX-2.
  • the ratio of COX-1 activity versus COX-2 activity for a COX-2 selective substrate is about 50% or less; in certain embodiments, 40% or less; in certain embodiments, 30% or less; in certain embodiments, 25% or less; in certain embodiments, 20% or less; in certain embodiments, 10% or less; in certain embodiments, 5% or less; in certain embodiments, 3% or less; in certain embodiments, 2% or less; and in certain preferred embodiments 1% or less.
  • a highly preferred COX-2 selective substrate is metabolized by COX-2, but is not metabolized by COX- 1.
  • COX-2 specific substrate and “COX-2 selective substrate” are used interchangeably herein.
  • enzyme activity refers to the rate at which substrate is consumed or product is formed in an enzymatic reaction under a given set of reaction conditions.
  • the Standard International (SI) unit for enzyme activity is an enzyme unit (U) and is defined as the amount of enzyme needed to produce 1
  • a unit may be defined differently herein [e.g., the amount of enzyme needed to produce 1 nmoles product per minute or the amount of enzyme needed to consume 1 ⁇ mole substrate per minute). Additional determinations of enzyme activity can be compared when utilizing similar or preferably identical reaction conditions. It is understood that reaction conditions can be changed and a new enzyme activity scale determined (e.g., by generating a standard curve of enzyme activity and use thereof, a process which is known to one of ordinary skill in the art).
  • the specific activity of a particular enzyme preparation refers to the total enzyme units divided by the total amount of protein present in the preparation. A preferred unit of specific activity is U per mg of protein (U/mg).
  • references to COX include both COX- 1 and COX-2.
  • Arachidonyl ethanolamide is defined herein to be a COX-2 selective substrate.
  • prostaglandin ethanolamides are included in the meaning of COX-2 selective metabolites.
  • cyclooxygenase COX
  • COX-1 COX-1
  • COX-2 cyclooxygenase-2
  • PG prostaglandin
  • PGs ethanolamide
  • EA prostaglandin ethanolamide
  • AEA arachidonyl ethanolamide
  • PG-EA prostaglandin-ethanolamide
  • Tx thromboxane
  • PG- EAs can include, but are not limited to: PGB2-EA, PGD 2 -EA,
  • PGE 2 -EA PGF 2 ⁇ -EA, TxB 2 -EA, 6-keto-PGF ⁇ -EA, 15-keto-PGE 2 - EA, 13, 14-dihydro-15-keto-PGE 2 -EA, PGG 2 -EA, PGH2-EA, PGA 2 - EA, PGJ2-EA, PGJ2-EA derivatives, bicyclo-PGE 2 -EA, TxA 2 -EA and PGI2-EA (also referred to herein as prostacyclin-EA) .
  • PGI2-EA also referred to herein as prostacyclin-EA
  • Tumor type typically references the tissue of tumor origin, but can also refer to the current tissue in which a tumor is located (e.g., colon cancer, liver cancer, or pancreatic cancer).
  • the stage and grade of a tumor is related to severity and medical definitions of stages and grades within a continuum are known in the art for each tumor or cancer type.
  • Each specialty within oncology e.g., hematology, colorectal, liver, pancreatic, lung, brain, dermatology, etc.
  • Tumor grade is determined by the appearance of the tumor under the microscope and how quickly the tumor is likely to grow and spread.
  • grading systems are different for each type of cancer, but are known to one of ordinary skill in the art.
  • grade I tumors are the least malignant appearing
  • grade II tumors are moderately differentiated with a moderately malignant appearance
  • grade III tumors are less differentiated and show enhanced signs of tissue invasion
  • grade IV tumors display the least differentiation and are the most malignant appearing.
  • the grade of a tumor is determined by one of ordinary skill in the art.
  • the stage of a tumor refers to the extent of a cancer, how advanced the tumor is in the patient (e.g., whether the disease has spread from the original site to other parts of the body) .
  • stage of a tumor is generally determined by radiographic studies such as a computed tomography (CT) scan, magnetic resonance (MRI) imaging and/ or ultrasound.
  • CT computed tomography
  • MRI magnetic resonance
  • Tumor staging is determined by one of ordinary skill in the art and can vary by tumor type or as a field advances, standard staging practices may change. Certain definitions of stages for various cancers are provided in the Dictionary of Cancer Terms on CANCERNET which is a service of the National Cancer Institute available on the World Wide Web at "http:/ /cancernet.nci.nih.gov/dictionary/ dictionaryfull.html", incorporated herein by reference in its entirety.
  • a physical location for inquiry or obtaining a copy of the Dictionary of Cancer Terms is: NCI Public Inquiries Office; Building 31, Room 10A03; 31 Center Drive, MSC 2580; Bethesda, MD 20892-2580. Staging refers to performing exams and tests to learn the extent of the cancer within the body, especially whether the disease has spread from the original site to other parts of the body.
  • Prostaglandins produced as a result of the activity of COX are known to have numerous physiological functions. These functions include the antithrombogenic action of prostacyclin released by the vascular endothelium and the cytoprotective effect of prostaglandins produced by the gastric mucosa (Whittle, et al. 1980, Nature, 284:271-273).
  • COX-2 is typically expressed following the activation of normal cells and certain atypically proliferating cells, by various pro-inflammatory agents including certain cytokines (Hla, T. and Nielson, K. 1992, Proc. Natl. Acad. Sci. USA, 89:7384-7388; Feng, et al. 1993, Arch.
  • Prostaglandins represent a class of substances produced in a wide variety of cells. In general, PGs act on the cells that produce them, on neighboring cells, or over short distances and can be classified as autocrine hormones. PGs and their relatives are usually thought of as potent local hormones (autocrine and paracrine) acting over a short lifetime. PGs, and related compounds, prostacyclin (PGI2), thromboxanes (TX), leukotrienes (LT), and lipoxins (LP), derive from fatty acids stored in cellular membranes as phospholipids or triglycerides, especially arachidonic acid, with an open chain, 20-carbon structure.
  • PGI2 prostacyclin
  • TX thromboxanes
  • LT leukotrienes
  • LP lipoxins
  • Prostaglandins generally resemble hairpins structurally with a five-membered ring and two chains extending from the ring. In general, substituents on the five- membered ring determine the subclass and activity of the prostaglandins. A series of synthetic reactions catalyzed by enzymes in the membranes, and certain non-enzymatic transformations, culminate in the release of prostaglandin product.
  • AEA is an unique substrate specific for COX-2.
  • COX-2 catalyzes the conversion of AEA to prostaglandin H2 ethanolamide (PGH 2 -EA).
  • PGH 2 -EA prostaglandin H2 ethanolamide
  • the PGH 2 -EA is subsequently enzymatically and nonenzymatically metabolized to a variety of compounds, such
  • PGE2-EA prostaglandin E2 ethanolamide
  • PPD2-EA prostaglandin D2 ethanolamide
  • PGH2-EA metabolites include, but are not limited to, PGB2-EA, PGD 2 -EA, PGE2-EA, PGF 2 ⁇ -EA, TxB 2 -EA, 6-keto-PGF ⁇ -EA, 15-keto-PGE 2 - EA, 13, 14-dihydro-15-keto-PGE 2 -EA, PGG2-EA, PGH2-EA, PGA 2 - EA, PGJ2-EA, PGJ2-EA derivatives, bicyclo-PGE -EA, HETEs, TXA2-EA and PGI2-EA., which are metabolized directly from AEA by COX-2, will also be referred to as PGH2-EA metabolites in this application.
  • prostaglandin and thromboxane ethanolamides are susceptible to enzymatic oxidation of the ethanolamide moiety and can undergo oxidation.
  • the amount of PGH 2 -EA metabolites in a biological sample correlates with the COX-2 specific activity in the subject from which the sample was collected.
  • the amount of COX-2 activity is a marker of or a measurement of inflammatory or cancerous disease processes.
  • Certain embodiments of the present invention include methods and materials for making sensitive measurements of picogram quantities of PGH2-EA metabolites. Further embodiments of the present invention include using these measurements of COX-2 activity to clinically grade or stage a disease process and to assess therapeutic outcomes.
  • a lipoamino acid is the substrate for COX-2.
  • the lipoamino acid is NAGly, which COX-2 catalyzes into PGH 2 -Gly and HETE-Gly.
  • detecting includes determining if a substance or compound is present in a sample.
  • the substance or compound being detected is preferably a PGH2-Gly or HETE-Gly metabolite.
  • Detecting can include measuring.
  • measuring means determining the relative or absolute amount of a substance or compound detected. Measurement is generally, but not always, performed relative to a standard. For example, the amount of the standard may be correlated with an amount of COX-2 activity or expression. Therefore, comparing the amount of metabolites measured in a sample from a subject indicates an amount of COX-2 activity or expression in the subject.
  • the metabolites may be detected and measured in a variety of ways, examples of which are discussed in greater detail below.
  • a sample is collected from a subject, and a selective COX-2 substrate of the present invention, such as NAGly, is added. Then, a corresponding metabolite is measured.
  • downstream metabolites may be measured.
  • both a COX-1 substrate such as arachidonic acid
  • a COX-2 selective substrate such as NAGly
  • the samples can be incubated with the substrates over time and a series of measurements of metabolites taken and compared in relation to the amount of time that passed.
  • Samples from the subject can be processed is several ways.
  • the sample may be extracted at least one time with a solvent, to remove the metabolites from the sample for analysis. Extraction can be followed by evaporation. The resulting residue may be redissolved in another solvent and analyzed. This process might involve several rounds of the extraction, evaporation and redissolving steps.
  • the solution resulting from one or more extractions of the sample may be filtered and analyzed.
  • the sample is extracted, filtered and analyzed for metabolite content.
  • One aspect of the present invention is a method of detecting COX-2 activity in a biological sample, comprising: incubation of the biological sample with NAGly, extracting the sample with a solvent, evaporating the solvent to leave a residue and analyzing the residue for metabolites wherein the presence of metabolites is indicative of COX-2 activity.
  • the amount of metabolites is measured and related to the quantity of COX-2, COX-2 expression, or COX-2 activity.
  • the analysis of metabolites includes, but is not limited to, detection by liquid chromatography- mass spectrometry.
  • a detection device for detecting PG metabolites of the present invention includes, but is not limited to, a mass spectrometer, a chromatography-coupled mass spectrometer, an immunoassay or an enzyme-linked immunoassay, or other means for detecting metabolites known in the art.
  • a mass spectrometer a mass spectrometer
  • chromatography-coupled mass spectrometer an immunoassay or an enzyme-linked immunoassay, or other means for detecting metabolites known in the art.
  • an immunoassay or an enzyme-linked immunoassay or other means for detecting metabolites known in the art.
  • liquid for example, liquid
  • chromatography/mass spectrometry (LC/MS) is conducted, preferably with a Waters 2690 Separations Module with a Zorbax RX-C18 narrow bore column (15 cm x 2.1 mm, 5 ⁇ m) interfaced to a Finnigan TSQ-7000 triple quadrupole mass spectrometer. Sodiated analytes are eluted with increasing concentrations of MeCN in 0.001% aqueous sodium acetate. Evaluation of PG metabolites in biological samples may be conducted with selected ion monitoring and quantification.
  • separation devices include, but are not limited to, extraction columns, affinity columns, filters, thin-layer chromatography plates and gels.
  • a PGH2- EA metabolite in a sample may be isolated or purified (separated partially or substantially from the natural constituents of a metabolite containing sample) using one or more techniques known in the art for the separation of chemical and especially prostaglandin compounds, for example, but not limited, to liquid chromatography.
  • the subject includes a mammal, such as a rodent, preferably a human, or a cultured cell of a mammal, including a cultured cell of a human.
  • a mammal such as a rodent, preferably a human, or a cultured cell of a mammal, including a cultured cell of a human.
  • Other subjects include farm animals and show animals (horses, cattle, sheep, pigs and swine, goats, fowl, and the like), pets (dogs, cats, parrots, canaries and the like),
  • 11672N:021089:498744:1:NASHVILLE animals kept in zoos and endangered species (elephants, lions, tigers, antelope, zebra, anteaters, water buffalo, pandas, cheetahs, kangaroos, ostriches, eagles, condors, finches, and the like) or a cultured cell of said animal.
  • endangered species elephants, lions, tigers, antelope, zebra, anteaters, water buffalo, pandas, cheetahs, kangaroos, ostriches, eagles, condors, finches, and the like
  • the sample is urine or may be collected from or processed from urine.
  • PG metabolites can be measured in urine by isotope dilution mass spectrometry.
  • the PG metabolites are generally stable in cell culture, bovine, canine and human CSF and human and rat plasma incubated about 5 hours at about 37°C.
  • a plurality of samples may be collected from the subject, with a period of time being allowed to pass between consecutive collections of the samples.
  • the amounts of PGH2-EA metabolites present in these samples are measured, compared and related to the periods of time that had been allowed to passed between collections.
  • the subject is a patient and the samples are taken in order to evaluate the effectiveness of anti-cancer therapy and to evaluate tumor state, severity or load in the patient.
  • samples can be collected from or prepared from cultured cells.
  • a variety of cells lines known to one skilled in the art are acceptable.
  • primary cell cultures can be used. Methods for collecting and culturing primary cell cultures are well known in the art.
  • Monoclonal and polyclonal antibodies to PGH2-EA metabolites or their metabolites can be made using standard antibody generation techniques in light of the present invention (Cohen, et al., U.S. Patent Serial No. 5,589,575, herein incorporated by reference; McCafferty, et al. 1996, Antibody Engineering, a Practical Approach, IRL Press; Mernaugh & Mernaugh 1994, Methods for the Production of Monoclonal Antibodies, in Molecular Methods in Plant Pathology).
  • monoclonal antibodies against PGE2 are commercially available from Cayman Chemical (118 E.
  • a wide variety of human diseases are associated with inflammation. These range from acute appendicitis to asthma, myocardial infarction, specific immunological disease processes, infection with viruses or bacteria, malignancy and metastasis, endotoxemia and reperfusion injury. Since COX-2 activity is important in the progression of these diseases, the present invention is a useful method of diagnosing or monitoring disease state. The present invention is also useful in detecting and treating non-malignant or immunological-related cell- proliferative diseases such as psoriasis, pemphigus vulgaris, Behcet's syndrome, acute respiratory distress syndrome (ARDS), ischemic heart disease, post-dialysis syndrome, leukemia, acquired immune deficiency syndrome, septic shock and other
  • the present invention can be used to facilitate the detection, measurement and treatment of any disorder which is etiologically linked to the inflammatory process.
  • a sample is collected from a patient suspected of having an inflammatory disease or diagnosed with an inflammatory disease and a metabolite of NAGly in the sample is detected and measured.
  • the amount of the metabolite present in the sample correlates with COX-2 activity in the patient and is a marker of the progress or severity of that disease or disease process.
  • a series of samples are collected from the patient over a period of time.
  • COX-2 expression is a marker for the metastatic
  • COX-2 expression was determined in 76 patients with a variety of stages of colorectal cancer (Sheehan, KM, et al, 1999, JAMA 282: 1254). Such studies can be used to generate a standard curve for COX-2 expression in cancer and colon cancer in particular (see supra).
  • Certain aspects of the present invention include methods of detecting a tumor in a patient in need thereof, comprising: obtaining a sample of the patient and detecting at least one metabolite of the present invention in the sample.
  • the presence of the metabolite in the sample is a marker for the presence of the tumor in the patient. More preferably, an amount of metabolite detected in the sample of the patient will be measured, wherein the amount of metabolite measured is indicative of the amount or severity of tumor present in the patient.
  • a further aspect of the present invention is a method of measuring and monitoring the size, grade, and/ or stage of a tumor, comprising: collecting a first sample of a subject and
  • 11672N:021089:498744:1:NASHVI LE measuring the amount of metabolites of the present invention in the first sample. Then a period of time is allowed to pass, during which the subject may, or may not, undergo anti-cancer therapy. After the period of time has passed, a second sample is collected from the subject and the amount of metabolites in the second sample is measured. The amounts of the metabolites in the first and second samples are compared, wherein the difference between the amounts of metabolites in the two samples is indicative of changes in the metabolism of the cancer cell.
  • a plurality of samples may be collected from the subject, with a period of time being allowed to pass between consecutive collections of the samples. The amounts of metabolites present in these samples are measured, compared and related to the periods of time that had been allowed to pass between collections.
  • the subject is a patient and the samples are collected in order to evaluate the effectiveness of anti-cancer therapy and to evaluate tumor severity or tumor load in the patient.
  • the effectiveness of the anti-cancer therapy is evaluated based on the changes in the amount of sample metabolite observed over time. For example, increases in the amount of metabolites over time indicate continued tumor growth and failure therapeutic intervention; whereas decreases in the amount of metabolites over time are indicative of therapeutic success and tumor regression.
  • the sample is a culture of cancer cells used as an experimental model or a culture of cancer cells taken from a patient.
  • the cultured cancer cells may
  • this procedure is done in order to determine an optimal anti-cancer therapy for that individual patient.
  • the attending health professional may characterize both an inflammatory process and a malignancy in the subject by detecting or measuring an amount of a prostaglandin ethanolamide in a sample of a subject specifically produced by the offending malignancy and inflammatory lesion.
  • Certain embodiments of the present invention provide an article of manufacture for the detection and /or measurement of COX-2 activity by the detection and/ or measurement of PGH2- EA or PGH2-Gly metabolites by radioassay or immunoassay, comprising an antibody and a set of instructions delineating a process for relating a detection and/ or measurement of PG metabolites in a sample to a detection and/ or measurement of COX-2 in a subject or a sample thereof.
  • the article of manufacture further comprises an antibody against the metabolites. More preferably, the article of manufacture further comprises the standard reagents required to perform an immunoassay, such as buffers, multi-well plates, additional antibodies and the like.
  • the article of manufacture further comprises one or more solid phase extraction columns for the isolation/ purification of the metabolites.
  • the article of manufacture further comprises a set of standards. More preferably, the article of manufacture further comprises an unlabeled PG metabolite internal standard for standard curve development.
  • the present invention provides an article of manufacture for the detection and/ or measurement of COX-2 activity by mass spectrometry, which comprises: a set of instructions delineating a process for relating a detection and/ or measurement of PG metabolites in a sample to a detection and/ or measurement of COX-2 in a subject or a sample thereof and a C18 solid phase extraction column.
  • the article of manufacture further comprises a set of standards. More preferably, the article of manufacture further comprises an unlabeled PG metabolite positive control, and a tetradeutereated PG metabolite internal standard.
  • the present invention establishes a role for COX-2 in the metabolism and regulation of an anti- inflammatory lipoamino acid, such as NAGly, .
  • COX-2 oxygenates NAGly with a moderate efficiency, in contrast to absence of metabolism by COX-1.
  • ranking the catalytic efficiency of COX-2 substrates from the most to the least efficient suggests that modification of the alcohol group in AEA to the carboxylic acid in NAGly, promotes higher affinity and more productive binding for catalysis by COX-2 [12,22].
  • the products identified here from NAGly oxygenation are similar to those obtained from the COX-2 metabolism of other substrates [1 , 11, 12].
  • NAGly is the first charged COX-2 selective substrate identified [11, 12] (Fig. 1). The highest levels of NAGly are observed in tissues that can be
  • NAGly metabolism by COX-2 may represent a mechanism for inactivation of this anti- inflammatory lipoamino acid, similar to the role of fatty acid amide hydrolase [17].
  • the anti-inflammatory and analgesic properties of NAGly are similar to many NSAIDs that would inhibit the COX-2 metabolism of NAGly [2,6- 9, 16, 17]. It is possible that COX-2 inhibition reduces pro- inflammatory PG production, and effectively raises the levels of anti-inflammatory lipid mediators such as NAGly and the endocannabinoids.
  • a sample may be urine or may be collected from or processed from urine.
  • PGH2-Gly metabolites can be measured in urine by isotope dilution mass spectrometry.
  • a fixed volume of urine is treated with an appropriate internal standard and then loaded on reversed-phase extraction cartridges.
  • the sample is then washed [e.g., 1 ml pH 4.0 20 mM Sodium acetate) and PG metabolites including the added internal standard are eluted with organic solvent [e.g., two 1 ml aliquots of MeCN). The solvent is evaporated and the residue is analyzed by LC/MS.
  • COX-2 expression and activity are generally linked with the inflammatory process, which accompanies a plethora of pathologies including, but not limited to, arthritis/ arthropathy, infectious disease, neurodegenerative disease, neoplasia and autoimmune disease.
  • the quantification of prostaglandin PGH2- Gly metabolites from biological fluids obtained non-invasively [e.g., blood, urine) will allow for the assessment of COX-2 activity in vivo, reflecting both inflammation and disease severity.
  • serial testing will allow for the tracking of the natural course of the disease as well as the efficacy of anti- inflammatory therapy.
  • a model for this application would be the ubiquitous use of C-reactive protein (CRP) in the diagnosis and assessment of diseases associated with inflammation.
  • CRP C-reactive protein
  • PGH2-Gly metabolites in this context instead of more traditional diagnostic markers, such as CRP, involve the highly specific nature of PGH2-Gly metabolite production. PGH2- Gly metabolites are elevated only when COX-2 activity is elevated whereas CRP elevations, for example, are very non- ' specific.
  • COX-2 expression and activity are linked to several solid tumors, most notably colorectal adenocarcinoma.
  • the quantification of PGH2-Gly metabolites from biological fluids, described herein, provide a noninvasive "early-warning" for clinically undetectable neoplasia.
  • serial testing following diagnosis will allow for the tracking of the natural course of the cancer as well as the efficacy of antineoplastic therapy.
  • a model for this application would be the use of prostate specific antigen (PSA) in the diagnosis and assessment of prostate adenocarcinomas.
  • PSA prostate specific antigen
  • PGH2-Gly metabolite quantification includes (a) relative noninvasiveness, (b) sensitivity (most cancers are advanced once symptomatic) and (c) cost (simple lab diagnostic technique versus colonoscopy for example) .
  • the present invention can be employed to identify a previously undescribed small molecule modulator of COX-2 activity. This can be done with the following method. Briefly, RAW264.7 cells at 30-40% confluence are activated with lipopolysaccharide (LPS, 20 ng/mL) and treated with a series of concentrations of a test compound. Cells are incubated for 12 h at 37°C and then medium is removed and replaced with buffered saline. Cells are then treated with 50 ⁇ M NAGly and incubated an additional 30 min at 37°C. Fpllowing incubation, buffered saline is collected and treated with tetradeutereated PGH2-EA metabolite standard.
  • LPS lipopolysaccharide
  • Buffered saline is extracted twice with equal volumes of 2: 1 CHCi3:MeOH.
  • the combined organic extract is evaporated under a stream of argon.
  • Quantisation of COX-2 activity is accomplished by comparing the area of the PGH2-Gly metabolite peak to that of the internal standard.
  • This example compares steady-state kinetic parameters for COX-2 mediated oxygenation of AA and NAGly.
  • N-Arachidonylglycine was purchased from Cayman
  • COX-1 was purified from ram seminal vesicles as previously described [19]. Site-directed mutagenesis of COX-2 was performed as described previously [20]. Recombinant COX-2 enzymes were expressed in Sf-9 insect cells and purified by ion-exchange chromatography and gel filtration as previously described [20]. Apo-enzymes were reconstituted with hematin prior to activity assays.
  • Mass Spectrometry Reactions for product characterization by mass spectrometry (MS) were performed in 100 mM Tris-HCl buffer containing 500 ⁇ M phenol, by incubating 86 ⁇ g COX-2 with 20 ⁇ g of NAGly for 2 min at 37°C. Reactions were quenched with 3 volumes of ice-cold methanol with or without triphenylphosphine (1 mg/ml). Samples were centrifuged at 14,000 rpms for 10 min at 4°C. Aliquots were removed and dried to completeness under a stream of argon. Samples were prepared for mass spectral analysis by reconstitution in 0.2% aqueous acetic acid solution and purification on a Waters Oasis HLB solid phase extraction
  • NAGly was built into the protein coordinates of uninhibited COX-2 (Protein Data Bank code 5COX). All amino acid positions were fixed except for the side chains of Arg-120, Arg-513, Glu-524, and Tyr-355.
  • the carboxylic acid of NAGly was restrained within 3.6A from the hydrogen bond donor/ acceptor groups of Arg-513 and Glu-524, while the amide carbonyl was restrained within 3.6A of Arg-120.
  • the Tyr-385 hydroxyl group was restrained within 3.6A from NAGly carbon C13 to ensure a productive conformation for oxygenation.
  • the complex was energy minimized for 1000 iterations using a conjugate gradient in the consistent valence forcefield.
  • Molecular dynamic simulations were then run on the energy minimized assemblies for 1000 iterations at 300K. All simulations were performed using the Discover module of Insight II 2000 with a R 12000 Silicon Graphics Octane workstation.
  • NAG Comparing kcat/Km values reported for the other selective substrates NAGly is 4-fold less efficient than 2-AG with a kcat/Km of 2.3 s-1 ⁇ M-1, but 8-fold more efficient than AEA with a kcat/Km of 0.065 s-1 ⁇ M-1 [12,22].
  • NAGly oxygenation was probed by site-directed mutagenesis of mCOX-2.
  • the active site tyrosyl radical formed at Tyr-385 initiates substrate oxygenation by 13- pro-S hydrogen abstraction, and when mutated to Y385F fails to oxidize neither AA nor NAGly [2].
  • AA binds in the COX active site with the f-end near Gly-533 [20,27].
  • the G533V mutant was used to incorporate steric hindrance near the Tf-end of the substrate channel to test the L-shaped conformation of NAGly. As expected no O2 uptake was observed after NAGly incubation with G533V.
  • 11672N:021089:498744:1:NASHVILLE displayed a modestly reduced capacity for AA oxygenation ( ⁇ 3- fold reductions) (Fig. 4).
  • the R120Q and E524L mutants exhibited a 6- and 7-fold reduction in the initial rate of NAGly oxygenation, when compared with wild-type (Fig. 4).
  • the Y355F mutant was more active towards NAGly, than AA (Fig. 4).
  • the side pocket of COX-2 is an extra solvent accessible space in the active site that contributes to the selectivity of inhibitors and substrates [2, 12].
  • Previous studies have identified residues in the side pocket of COX-2, especially Arg- 513, as critical determinants of 2-AG oxygenation by COX-2 [13]. Mutantions that change COX-2 side pocket residues (Val- 434, Arg-513, and Val-523) to their corresponding amino acids in COX-1 (Ile-434, His-513, and Ile-523), were tested for effects on NAGly oxygenation. As previously reported, the mutations did not greatly affect the COX-2 ability to metabolize AA [13] (Fig. 5).
  • the V523I mutation modestly reduced the initial rate of NAGly metabolism to 50% of that observed with wild-type enzyme.
  • the R513H mutation markedly reduced the NAGly oxygenation rate, and the double mutant R513H/V523I was indistinguishable from R513H alone (Fig. 5).
  • All enzymes that contained histidine at position 513, including the triple mutant V434I/R513H/V523I (VRV) and oCOX-1 displayed a reduced ability to oxygenate NAGly (Fig. 5). These results are similar to those obtained with 2-AG, and thus it appears that Arg-513 dictates in part, the isoform selectivity of the novel substrate NAGly [13].

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Abstract

L'invention concerne une méthode pour détecter une activité d'une enzyme, la COX-2, chez un sujet, ladite méthode consistant à : prélever un échantillon chez le sujet ; détecter la présence d'un métabolite d'eicosanoïdes d'acides aminés dans l'échantillon, la présence d'un tel métabolite indiquant l'activité de la COX-2 du sujet. De préférence, le métabolite d'eicosanoïdes d'acides aminés est un métabolite de PGH2-Gly ou HETE-Gly. Ce métabolite peut être détecté sur la base du métabolisme d'un substrat sélectif de la COX-2. De préférence, ce substrat est un acide lipoaminé. Mieux encore, l'acide lipoaminé est sélectionné parmi la NAGly, la N-arachidonyl-alanine, et l'acide δ-arachidonyl-aminobutuyique.
PCT/US2002/024864 2001-08-07 2002-08-06 Compositions et methodes pour detecter et quantifier l'activite de la cox-2 par le metabolisme d'acides lipoamines Ceased WO2003014699A2 (fr)

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Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CROMLISH, W.A. ET AL.: 'High-Level expression of Active Human cyclooxygenase-2 in Insect Cells' ARCH.BIOCHEM.BIOPHYS vol. 314, no. 1, October 1994, pages 193 - 199, XP002984878 *
MITCHELL, J.A. ET AL.: 'Induction of Cyclo-Oxygenase-2 by Cytokines in Human Pulmanry Epithelial Cells: Regulation by Dexamethasone' BR.J.PHARMACOL vol. 113, no. 3, November 1994, pages 1008 - 1114, XP008044617 *
SHAPPELL, S.B. ET AL.: 'Alterations in Lipoxygenase and Cyclooxygenase-2 Catalytic Activity and mRNA Expression in Prostate Carcinoma' NEOPLASIA vol. 3, no. 4, July 2001 - August 2001, pages 287 - 303, XP008044620 *

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