Detection of glucuronidated and 3-bromotyrosine

Abstract

The present invention to provides methods, kits, and compositions for: i) detecting the level of 3-bromotyrosine in a sample that has been treated to liberate 3-bromotyrosine from 4-O-glucuronide-3-bromotyrosine, and/or ii) detecting the level of 4-O-glucuronide-3-bromotyrosine, and/or the combined level of both 4-O-glucuronide-3-bromotyrosine and 3-bromotyrosine, in a sample that has not been treated to liberate 3-bromotyrosine from 4-O-glucuronide-3-bromotyrosine. In certain embodiments, such detected levels are used to: i) identify the presence, severity, or risk of an eosinophilic disorder (e.g., asthma or a TH2-high eosinophilic disorder); ii) identify therapy effective for treating asthma or an eosinophilic disorder; or iii) identify patients suitable for treatment with therapeutic agents targeted to asthma or an eosinophilic disorder.

Claims

1. A method of testing a sample comprising: testing a sample from a human subject to determine the level of 3-bromotyrosine, wherein said sample has been treated to liberate 3-bromotyrosine from 4-O-glucuronide-3-bromotyrosine; and wherein said sample is selected from the group consisting of: urine, saliva, serum, plasma, tissue biopsy, and whole blood.

2. The method of claim 1, wherein said sample is from a human subject with, or suspected of having, asthma.

3. The method of claim 1, wherein said sample has been treated with a de-glucuronidation agent and/or procedure.

4. The method of claim 3, wherein said de-glucuronidating agent comprises β-glucuronidase.

5. The method of claim 1, wherein said testing said sample comprises performing a detection method selected from the group consisting of: mass spectrometry, an immunological assay, HPLC-UV/VIS, LC-Electrochemical detection, or LC-Ampimetric detection method.

6. The method of claim 1, wherein said sample comprises urine.

7. A method of identifying the presence, severity, risk of, or risk of exacerbation of asthma in a human subject comprising: testing a sample from a human subject to determine the level of 3-bromotyrosine, wherein said sample has been treated to liberate 3-bromotyrosine from 4-O-glucuronide-3-bromotyrosine, and wherein said sample is selected from the group consisting of: urine, saliva, serum, plasma, tissue biopsy, and whole blood; b) identifying the presence, severity, risk of, or risk of exacerbation of asthma in said subject based on: i) an elevated level of 4-O-glucuronide-3-bromotyrosine in said sample compared to a control; and/or ii) an elevated combined level of 4-O-glucuronide-3-bromotyrosine and 3-bromotyrosine in said sample compared to a control.

8. The method of claim 7, wherein the presence of asthma is identified in said subject.

9. The method of claim 7, wherein the severity of asthma is identified in said subject.

10. The method of claim 7, wherein said subject, prior to step a) is identified as having or being at risk of asthma.

11. The method of claim 7, further comprising: c) treating said subject with a therapeutic agent used to treat asthma.

12. The method of claim 7, further comprising: c) generating, transmitting, and/or graphically displaying a report that indicates said 4-O-glucuronide-3-bromotyrosine from said second sample, and/or said combined level of both 4-O-glucuronide-3-bromotyrosine and 3-bromotyrosine from said sample, are elevated compared to corresponding controls, and/or that said subject is in need of a therapeutic agent used to treat asthma.

13. The method of claim 7, further comprising: c) generating, transmitting, and/or graphically displaying a report that indicates said 4-O-glucuronide-3-bromotyrosine from said sample, and/or said combined level of both 4-O-glucuronide-3-bromotyrosine and 3-bromotyrosine from said sample, are elevated compared to corresponding controls, and/or that said subject has or is at risk of asthma.

14. The method of claim 7, wherein said sample is from a human subject with, or suspected of having, asthma.

15. The method of claim 7, wherein said sample has been treated with a de-glucuronidation agent.

16. The method of claim 15, wherein said de-glucuronidation agent comprises β-glucuronidase.

17. The method of claim 7, wherein said testing said sample comprises performing a detection method selected from the group consisting of: mass spectrometry, an immunological assay, HPLC-UV/VIS, LC-Electrochemical detection, or LC-Ampimetric detection method.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1. The baseline distribution of Free and total BrTyr. The Box plot represent the median and interquartile range. Whiskers represent the 1st quartile−1.5*(interquartile range) and 3rd quartile+1.5*(interquartile range). Note that free (non-glucurinidated) BrTyr represents only a small fraction of the total BrTyr (˜5%). Moreover, there is large variation in how much glucurinidated BrTyr there is.

(2) FIG. 2. Correlation between Free and “Total” BrTyr. Total BrTyr is generated by glucuronidase enzyme digestion treatment. Only 17.9% of variation in free BrTyr levels are accounted for by variations in Total BrTyr. Thus, while there exists a modest statistical correlation, the two measures are fundamentally independent of one another.

(3) FIG. 3. Free (Panel A) and Total (Panel B) BrTyr baseline levels of asthmatics that increase their asthma control test with 3 or more points after 3 months of mepolizumab. Asthmatics that show improvement in quantitative assessments of symptoms (increase of their asthma control test by 3 or more points) following 3 months of mepolizumab therapy have higher levels of both Free (Panel A) and Total (Panel B) BrTyr at baseline. Those with the highest BrTyr levels, evidence of eosinophil activation and tissue injury, are most likely to show improvement in Asthma Control Test results (reduction in symptom severity on drug).

(4) FIGS. 4A-D. ORs and 95% Cl for baseline free BrTyr (ng/mg Cr) and a positive clinical outcome (3 point or greater asthma control test response) to mepolizumab. Results shown represent the ORs (filled circles) and 95% Cl (lines) Asterisk indicates P<0.05 as determined by likelihood-ratio c test. Free urinary BrTyr levels predict dose dependent risk for asthma presence and severity. And correspondingly, those with higher levels of urinary BrTyr at baseline are dose dependently more likely to show improvement in their asthma severity questionnaire (Asthma Control Test) following 3 months of mepolizumab therapy. FIG. 4A, BrTyr is stratified by above v below median value; FIG. 4B by tertiles; FIG. 4C by quartiles; and FIG. 4D BrTyr is stratified by ROC curve cut-point.

(5) FIGS. 5A-D. ORs and 95% Cl for baseline free BrTyr (ng/mg Cr) and a positive clinical outcome (3 point or greater asthma control test response) to mepolizumab. Results shown represent the ORs (filled circles) and 95% Cl (lines) Asterisk indicates P<0.05 as determined by likelihood-ratio c test. Because so much of the BrTyr is glucuronidated, after enzyme digestion and release, the level of Total BrTyr quantified is 10-20 fold higher than that of free BrTyr, and correspondingly, is much more accurately measured. Thus, one sees stronger and cleaner clinical signals when examining those with higher levels of urinary Total BrTyr at baseline. Total BrTyr predicts dose dependent improvement in asthma severity questionnaire (Asthma Control Test) in response to 3 months of mepolizumab therapy. FIG. 5A stratified by above v below median value. FIG. 5B by tertiles, FIG. 5C by quartiles, and FIG. 5D by ROC curve cut-point.

(6) FIG. 6. The baseline distribution of Free and total BrTyr. The Box plot represent the median and the 75 and 25 percentile cutoffs. The whiskers represent the 1st quartile−1.5*(interquartile range) and 3rd quartile+1.5*(interquartile range).

(7) FIG. 7. The correlation between Free and total BrTyr in the severe asthma cohort at baseline (before steroid withdraw).

(8) FIG. 8. Baseline Total BrTyr levels in asthma phenotypes.

(9) FIG. 9. ORs and 95% CI for the associations between Total BrTyr levels and unscheduled MD visits. Results shown represent the ORs (circles) and 95% CI (lines) of unscheduled MD visited and (A) high level of bromotyrosine (greater than median level of 2.25 ng/mg cr, filled circle) versus not a high level (open circle) and (B) high level of bromotyrosine (greater than <1.60 ng/mg Cr), filled circle) versus not a high level (open circle).

(10) FIG. 10 bromotyrosine metabolism and exemplary quantification method.

(11) FIG. 11 shows how β-glucuronidase catalyzes hydrolysis of 4-o-glucuronide-3-bromotyrosine.

(12) FIG. 12. Panel A shows 13C6 labeled 3-bromotyrosine. Panel B shows 13C6 labeled 4-O-glucuronide-3-bromotyrosine.

(13) FIG. 13 shows a scatter plot of urine free 3-bromotyrosine (BrY) after glucuronidase incubation in non-severe asthmatic patients (n=65, left panel) and severe asthmatic patients (n=120, right panel) at baseline vs one month's medication (40 mg injected Triamcinolone) with mean±SD indicated. BrY was quantified by LC/MS/MS with .sup.13C.sub.6-BrY as internal standard and expressed as per mg creatinine. P value was calculated by Wilcoxon Rank Sum test.

DEFINITIONS

(14) The terms “individual,” “host,” “subject,” and “patient” are used interchangeably herein, and generally refer to a mammal, including, but not limited to, primates, including simians and humans, equines (e.g., horses), canines (e.g., dogs), felines, various domesticated livestock (e.g., ungulates, such as swine, pigs, goats, sheep, and the like), as well as domesticated pets and animals maintained in zoos. In some embodiments, the subject is specifically a human subject.

DETAILED DESCRIPTION

(15) The present invention relates to methods, kits, and compositions for: i) detecting the level of 3-bromotyrosine in a sample that has been treated to liberate 3-bromotyrosine from 4-O-glucuronide-3-bromotyrosine, and/or ii) detecting the level of 4-O-glucuronide-3-bromotyrosine, and/or the combined level of both 4-O-glucuronide-3-bromotyrosine and 3-bromotyrosine, in a sample that has not been treated to liberate 3-bromotyrosine from 4-O-glucuronide-3-bromotyrosine. In certain embodiments, such detected levels are used to: i) identify the presence, severity, or risk of an eosinophilic disorder (e.g., asthma or a TH2-high eosinophilic disorder); ii) identify therapy effective for treating asthma or an eosinophilic disorder; or iii) identify patients suitable for treatment with therapeutic agents targeted to asthma or an eosinophilic disorder.

(16) Work conducted during development of embodiments described herein revealed that urinary free bromotyrosine, which for more than a decade has been used as a marker of eosinophil activation, represents only a minor component of the BrTyr formed during eosinophil activation. Most of the BrTyr is, in fact, metabolized and glucuronidated, forming 4-O-glucuronide-3-bromotyrosine. Consequently, only a small proportion (˜5%) of BrTyr is present in its free form in urine, while the majority is glucuronidated in urine. It was found that by liberating BrTyr via enzyme digestion, one can generate a signal that is, for example, about 20-fold higher, and generally easier to monitor analytically for measurement.

(17) Work conducted during the development of embodiment described herein found that bromotyrosine (BrTyr) (as shown in FIGS. 10-11) is metabolized into a glucuronidated form, 4-O-glucuronide-3-bromotyrosine (see FIGS. 10-11), whose level is much larger than free BrTyr, and which is highly variable and poorly correlated with free BrTyr. In certain embodiments, to get a true quantification of Total BrTyr, one can enzymatically digest (or otherwise treat) a sample (e.g., urine, saliva, plasma, serum, blood, etc.) with β-glucuronidase, to promote hydrolysis of the 4-O-glucuronide-3-bromotyrosine, releasing free BrTyr. Thus, in some embodiments, measurement of “Total” BrTyr in post de-glucuronidated (e.g., glucuronidase digested) samples yields a sum of the free BrTyr (i.e., naturally present as free BrTyr), plus all the glucuronidated BrTyr. The methods, systems, kits, and compositions herein are not limited by the agent or procedure used to de-glucuronidate 4-O-glucuronide-3-bromotyrosine to liberate free BrTyr. In certain embodiments, reagents and procedures are employed that do not employ acid, to avoid producing artifactual BrTyr, which can be generated under such acidic conditions.

(18) In work conducted during development of embodiments described herein, is was found that certain urine samples contained approximately 5% Free BrTyr (which is non-glucuronidated Bromotyrosine and is as shown in FIGS. 10-11), and that approximately 95% BrTyr is 4-O-glucuronide-3-bromotyrosine. Therefore, “total BrTyr” refers to naturally free BrTyr+glucuronidase (or other de-glucuronidation agent) treated 4-O-glucuronide-3-bromotyrosine. In certain embodiments, liberating 3-bromotyrosine from glucuronidated BrTyr via enzyme digestion (or other de-glucuronidation agent) provides Total Bromotyrosine whose signal is approximately 10-20-fold higher than free BrTyr (e.g., 10 times . . . 15 times . . . 18 times . . . 20 times . . . or 25 times higher). In certain embodiments, detection of 4-O-glucuronide-3-bromotyrosine or total BrTyr (e.g., in a de-gluroronidating agent treat sample), is employed to assess disease risk and/or monitor or design therapy for such diseases and conditions such as asthma, or a TH2-high eosinophil-rich disease, such as eosinophilic granulomatosis with polyangiitis (previously Churg-Strauss Disease), Eosinophilic Esophagitis, Eosinophilic Gastritis, Eosoniphilic Colitis, Eosinophilic Enteritis, Hypereosinophilic syndrome (blood or organ) or other eosinophilic proliferative disorders.

(19) The present invention is noted limited to any particular method to detect 4-O-glucuronide-3-bromotyrosine and 3-bromotyrosine. For example, 4-O-glucuronide-3-bromotyrosine and 3-bromotyrosine can be measured using any suitable methodology, including but not limited, to mass spectrometry, HPLC/UV, HPLC/Vis, LC/MS/MS, immunological detection methods. In other embodiments, 4-O-glucuronide-3-bromotyrosine and 3-bromotyrosine are measured using: 1) a sandwich immunoassay (e.g., monoclonal, polyclonal and/or DVD-Ig sandwich immunoassays or any variation thereof (e.g., monoclonal/DVD-Ig or DVD-Ig/polyclonal), including chemiluminescence detection, radioisotope detection (e.g., radioimmunoassay (MA)) and enzyme detection (e.g., enzyme immunoassay (EIA) or enzyme-linked immunosorbent assay (ELISA) (e.g., Quantikine ELISA assays, R&D Systems, Minneapolis, Minn.))), 2) a competitive inhibition immunoassay (e.g., forward and reverse), 3) a fluorescence polarization immunoassay (FPIA), 4) an enzyme multiplied immunoassay technique (EMIT), 5) a bioluminescence resonance energy transfer (BRET), 6) a homogeneous chemiluminescent assay, 7) a SELDI-based immunoassay, 8) chemiluminescent microparticle immunoassay (CMIA) and 9) a clinical chemistry colorimetric assay (e.g., IMA, creatinine for eGFR determination and LC-MS/MS). (See, e.g., Tietz Textbook of Clinical Chemistry and Molecular Diagnostics. 4th Edition, edited by C A Burtis, E R Ashwood and D E Bruns, Elsevier Saunders, St. Louis, Mo., 2006.).

(20) Further, if an immunoassay is being utilized, any suitable detectable label as is known in the art can be used. For example, the detectable label can be a radioactive label (such as 3H, 1251, 35S, 14C, 32P, and 33P), an enzymatic label (such as horseradish peroxidase, alkaline peroxidase, glucose 6-phosphate dehydrogenase, and the like), a chemiluminescent label (such as acridinium esters, thioesters, or sulfonamides; luminol, isoluminol, phenanthridinium esters, and the like), a fluorescent label (such as fluorescein (e.g., 5-fluorescein, 6-carboxyfluorescein, 3′6-carboxyfluorescein, 5(6)-carboxyfluorescein, 6-hexachloro-fluorescein, 6-tetrachlorofluorescein, fluorescein isothiocyanate, and the like)), rhodamine, phycobiliproteins, R-phycoerythrin, quantum dots (e.g., zinc sulfide-capped cadmium selenide), a thermometric label, or an immuno-polymerase chain reaction label. An introduction to labels, labeling procedures and detection of labels is found in Polak and Van Noorden, Introduction to Immunocytochemistry, 2nd ed., Springer Verlag, N.Y. (1997), and in Haugland, Handbook of Fluorescent Probes and Research Chemicals (1996), which is a combined handbook and catalogue published by Molecular Probes, Inc., Eugene, Oreg. A fluorescent label can be used in FPIA (see, e.g., U.S. Pat. Nos. 5,593,896, 5,573,904, 5,496,925, 5,359,093, and 5,352,803, which are hereby incorporated by reference in their entireties). An acridinium compound can be used as a detectable label in a homogeneous or heterogeneous chemiluminescent assay (see, e.g., Adamczyk et al., Bioorg. Med. Chem. Lett. 16: 1324-1328 (2006); Adamczyk et al., Bioorg. Med. Chem. Lett. 4: 2313-2317 (2004); Adamczyk et al., Biorg. Med. Chem. Lett. 14: 3917-3921 (2004); and Adamczyk et al., Org. Lett. 5: 3779-3782 (2003)).

(21) In certain embodiments, 4-O-glucuronide-3-bromotyrosine is detected with one antibody or antibody fragment that recognizes the 3-bromotyrosine moiety (e.g., as part of immunocapture), and then a glucose oxidase reaction is initiated to generate H202 from the 4-O-glucuronide moiety. A peroxidase coupled reagent is then employed to visualize the color reaction.

(22) In certain embodiments, 4-O-glucuronide-3-bromotyrosine and/or 3-bromotyrosine are detected by one of the following methods: i) HPLC with electrochemical detection; ii) HPLC with Ampimetric detection; iii) HPLC with UV/VIS detection; and iv) derivitization and GC/MS detection.

(23) In some embodiments, 4-O-glucuronide-3-bromotyrosine and/or 3-bromotyrosine are detected by methods employing mass spectrometry. Exemplary mass detection methods for 3-bromotyrosine are described in Example 1 below. 4-glucuronide-3-bromotyrosine can be monitored, for example, by LC/ESI/MS/MS in positive MRM mode directly with parent to daughter transitions: m/z 436>390, 436>311, 438>392, 438>311. In certain embodiments, stable isotope labeled 3-bromotyrosine molecules, and/or stable isotope labeled 4-O-glucuronide-3-bromotyrosine internal standards are employed (see, e.g., FIG. 12). In certain embodiments, 3-bromotyrosine is detected in urine employing methods described in the art (e.g., using LC/EST/MS/MS) as described in Wedes et al., Clin Transl Sci. 2009 April; 2(2):112-7 and Wedes et al., J Pediatr., 2011 August; 159(2):248-55.e1, both of which are herein incorporated by reference in their enteritis, specifically for the mass spectrometric detection methods described therein).

(24) In certain embodiments, as part of the methods of the present invention, a patient is administered or recommended for administration of a therapeutic agent. Examples of such agents include, for example: Mepolizumab/Nucala (GSK anti-IL5); Reslizumab (Cinquil™, Teva Pharmaceuticals; formerly SCH-55700 anti-IL5); Benralizumab (a humanized afucosylated IgG1K mAb to human IL-5Ra); Lebrikizumab (anti-IL-13, Genentech/Chugai Pharmaceutical); Tralokinumab (CAT-354, MedImmune; an injectable anti-IL-13 humanized IgG4 mAb); a recombinant human IL-4 variant called pitrakinra (Aerovant™, Aerovance; which competitively inhibits the IL-4Ra receptor complex to interfere with the actions of both IL-4 and IL-13); Dupilumab (SAR231893/REGN668; which is a fully humanized mAb to the IL-4Ra/IL-13Rα1 receptor complex that inhibits both IL-4 and IL-13 signaling); a leukotriene receptor antagonist (LTRA); a steroidal drug (e.g., as shown in Table 5); and a non-steroidal drug (e.g., as shown in Table 6). In certain embodiments, the therapeutic agent is a TH2-targeted biologic therapy, such as those targeting pathways of: IL4, IL5, IL13, IL17/25. In some embodiments, anti-IgE therapy is employed. Additional examples of therapeutic agents (e.g., for treating an eosinophilic disorder) include, but are not limited to: corticosteroids (e.g., prednisone), Flovent, and budesonide.

(25) TABLE-US-00001 TABLE 5 Steroidal Drugs Generic Name Chemical Name Brand Name Beclomethasone 9-chloro-11β,17,21-trihydroxy-16βmethylpregna-1,4- QVAR Dipropionate HFA diene-3,20-dione 17,21-dipropionate. Inhalation Aerosol 40 mcg/ puff & 80 mcg/ puff Budesonide 16,17-(butylidenebis(oxy))-11,21-dihydroxy-, (11- Plumicort β,16-α)-pregna-1,4-diene-3,20-dione Flexhaler & Plumicort Respules Budesonide in (RS)-11β,16α,17,21-Tetrahydroxypregna-1,4-diene- Symbicort combination with 3,20-dione cyclic 16,17-acetal with butyraldehyde Formoterol (Budesonide) (R*,R*)-(±)-N-[2-hydroxy-5-[1-hydroxy-2[[2-(4- methoxyphenyl)-1- methylethyl]amino]ethyl]phenyl]formamide,(E)-2- butendioate(2:1),dihydrate Ciclesonide 2-[(1S,2S,4R,8S,9S,11S,12S,13R)-6-cyclohexyl- Alvesco 11-hydroxy-9,13-dimethyl-16-oxo-5,7- Inhalation dioxapentacyclo[10.8.0.02,9.04,8.013,18]icosa-14, Aerosol 17-dien-8-yl]-2-oxoethyl 2-methylpropanoate Flunisolide (1S,2S,4R,8S,9S,11S,12S,13R,19S)-19-fluoro-11- Aerobid Aerosol hydroxy-8-(2-hydroxyacetyl)-6,6,9,13-tetramethyl-5,7- and Aerobid-M dioxapentacyclo[10.8.0.02,9.04,8.013,18]icosa-14,17- Aerosol dien-16-one Fluticasone S-(fluoromethyl)-6α,9-difluoro-11β,17-dihydroxy- Flovent HFA Propionate 16α-methyl-3-oxoandrosta-1,4-diene-17β- and Flovent carbothioate,17-propanoate Diskus Fluticasone in S-(fluoromethyl)-6α,9-difluoro-11β,17-dihydroxy- Advair Diskus & combination with 16α-methyl-3-oxoandrosta-1,4-diene-17β- Advair HFA Salmeterol carbothioate,17-propanoate [and] (RS)-2- (broncodialator) (hydroxymethyl)-4-{1-hydroxy-2-[6-(4-phenylbutoxy) hexylamino]ethyl}phenol Mometasone furoate (11β,16α)-9,21-dichloro-11-hydroxy-16-methyl-3,20- Asmanex dioxopregna-1,4-dien-17-yl 2-furoate Twisthaler Mometasone in (11β,16α)-9,21-dichloro-11-hydroxy-16-methyl-3,20- Dulera combination with dioxopregna-1,4-dien-17-yl 2-furoate [and] rac-(R,R)- Formoterol N-[2-hydroxy-5-[1-hydroxy-2-[1-(4-methoxyphenyl) (broncodialtor) propan-2-ylamino]ethyl]phenyl]formamide Triamcinolone (4aS,4bR,5S,6aS,6bS,9aR,10aS,10bS)-4b-fluoro-6b- Azmacort acetonide glycoloyl-5-hydroxy-4a,6a,8,8-tetramethyl- Inhalation 4a,4b,5,6,6a,6b,9a,10,10a,10b,11,12-dodecahydro-2H- Aerosol naphtho[2′,1′:4,5]indeno[1,2-d][1,3]dioxol-2-one Prednisone 17,21-dihydroxypregna-1,4-diene-3,11,20-trione Deltasone Prednisolone (11β)-11,17,21-trihydroxypregna-1,4-diene-3,20-dione Methylprednisolone (1S,2R,8S,10S,11S,14R,15S,17S)-14,17-dihydroxy- Medrol, Solu- 14-(2-hydroxyacety1)-2,8,15- Medrol, Depo- trimethyltetracyclo[8.7.0.02,7.011,15]heptadeca-3,6- Medrol dien-5-one Dexamethasone (8S,9R,10S,11S,13S,14S,16R,17R)-9-Fluoro-11,17- Decadron dihydroxy-17-(2-hydroxyacetyl)-10,13,16-trimethyl- 6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H- cyclopenta[a]phenanthren-3-one

(26) TABLE-US-00002 TABLE 6 Non-Steroidal Drugs Generic Name Chemical Name Brand Name Type Albuterol Sulfate α1 (tert-butylamino) methyl]-4-hydroxy-m- VoSpireER LABA - xylene-α,α′-diol sulfate (2:1) (salt) Extended recommended Release Tablets with use with steroids Formoterol ±)-2-hydroxy-5-[(1RS)-1-hydroxy-2-[[(1RS)-2- Foradil LABA - fumarate (4-methoxyphenyl)-1methylethyl]- Aerolizer recommended amino]ethyl]formanilide fumarate dihydrate with use with steroids Salmeterol ±-4-Hydroxy-α1-[[[6-(4- Serevent Diskus LABA - Xinafoate phenylbutoxy)hexyl]amino]methyl]-1,3- recommended benzenedimethanol compd. with 1-hydroxy-2- with use with naphthalenecalboxylic acid (1:1) steroids Albuterol Sulfate α1-(tertbutylamino) methyl]-4-hydroxy-m- ProAir HFA, SABA - rescue HFA xylene-α,α′-diol sulfate (2:1) (salt) Proventil HFA, medicine Ventolin HFA Albuterol Sulfate α1 [(tert-butylamino) methyl]-4-hydroxy-m- SABA - rescue Inhalation xylene-α,α′-diol sulfate (2:1) (salt) medicine Solution Albuterol Sulfate α1 [(tert-butylamino) methyl]-4-hydroxy- AccuNeb SABA - rescue Nebulizer mxylene-α,α′-diol sulfate (2:1) (salt) Inhalation medicine Soluction Solution, Albuterol Sulfate 0.5% Ipratropium [8-methyl-8-(1-methylethyl)-8- Combivent; Anticholinergic Bromide in azoniabicyclo[3.2.1]oct-3-yl] 3-hydroxy-2- DuoNeb combination with phenyl-propanoate [and] α1 [(tert-butylamino) Albuterol Sulfate methyl]-4-hydroxy-mxylene-α,α′-diol sulfate (2:1) (salt) Ipratropium [-methyl-8-(1-methylethyl)-8- Atrovent, Anticholinergic Bromide HFA azoniabicyclo[3.2.1]oct-3-yl] 3-hydroxy-2- Apovent and phenyl-propanoate Aerovent Levalbuterol HCl (R)-α1-[[(1,1-dimethylethyl)amino]methyl]-4- Xopenex; SABA - rescue hydroxy-1,3-benzenedimethanol hydrochloride Xopenex HFA medicine Pirbuterol (RS)-6-[2-(tert-butylamino)-1-hydroxyethyl]-2- Maxair SABA - rescue (hydroxymethyl)pyridin-3-ol Autoinhaler medicine Tiotropium (1α,2β,4β,5α,7β)-7-[(Hydroxydi-2- Spiriva SABA - rescue Bromide thienylacetyl)oxy]-9,9-dimethyl-3-oxa-9- HandiHaler medicine Inhalation azoniatricyclo[3.3.1.02,4]nonane bromide Powder monohydrate Terbutaline (RS)-5-[2-(tert-butylamino)-1- Brethine, SABA - rescue hydroxyethyl]benzene-1,3-diol Bricanyl, medicine Brethaire, Terbulin Cromolyn disodium 5,5′-[(2- Intal, Intal Non-steroid anti- Sodium hydroxytrimethylene)dioxy]bis[4-oxo-4H-1- Metered Dose inflammatory benzopyran-2-carboxylate] Inhaler Theophylline 1H-Purine-2,6-dione,3,7-dihydro,1,3-dimethyl- Uniphyl, Non-steroid anti- Elixophyllin, inflammatory Theo-24, Theo- Time, Theochron Montelukast (S,E)-2-(1-((1-(3-(2-(7-chloroquinolin-2- Singular, Leukotriene yl)vinyl)phenyl)-3-(2-(2-hydroxypropan-2- Montelo-10 receptor yl)phenyl)propylthio)methyl)cyclopropyl)acetic antagonist acid (LTRA) Zafirlukast cyclopentyl 3-{2-methoxy-4-[(o- Accolate, Leukotriene tolylsulfonyl)carbamoyl]benzyl}-1-methyl-1H- Accoleit, receptor indol-5-ylcarbamate Vanticon antagonist (LTRA) Zileuton N-[1-(1-benzothien-2-yl)ethyl]-N-hydroxyurea Zyflo, Zyflo CR Leukotriene receptor antagonist (LTRA) Omalizumab Accession Number DB00043 Xolair Humanized Antibody Dyphylline 7-(2,3-dihydroxypropyl)-theophylline Lufyllin Broncodilator Dyphylline in 7-(2,3-dihydroxypropyl)-theophylline [and] COPD; Lufyllin- Broncodilator and combination with (RS)-3-(2-methoxyphenoxy)propane-1,2-diol GG expectorant Guaifenesin

EXAMPLES

Example 1

(27) Since omalizumab was approved in 2003, biologic therapies for asthma have skyrocketed, with more than 30 drugs in clinical trials and many more in development. The goal is to tailor asthma biologic therapies to specific asthma phenotypes. Given the recent approval of anti-IL5 therapies (e.g., mepolizumab and reslizumab), it is clear that identifying underlying endotypes and clinical phenotypes is essential to assign appropriate therapy.

(28) Methods

(29) For Urine Free BrTyr:

(30) 200 ul urine was aliquoted into a glass tube with 20 ul 0.5 uM [.sup.13C.sub.6]-BrTyr and 1 mM [.sup.13C.sub.9, .sup.15N.sub.1]-tyrosine added as internal standard. The urine sample was diluted with 2 ml 0.1% formic acid in water and loaded to DSC-18 3 mL SPE column, previously balanced with 2×3 ml methanol and then 2×3 ml 0.1% formic acid in water. The column was washed with 2×3 ml 0.1% formic acid in water, then eluated with 2×3 ml 0.1% formic acid in 30% methanol. The elute was dried under SpeedVacuum and re-suspended in 100 ul H2O. Supernatants (5 μl) were analyzed by injection onto a Titan™ C18 UHPLC Column (1.9 μm particle size, L×I.D. 10 cm×2.1 mm, Supelco) at a flow rate of 0.4 ml min.sup.−1 using a 2 Shimadazu LC-20AD Nexera CL pump system, SIL-30AC MP CL autosampler interfaced with an Shimadzu 8050 mass spectrometer. A discontinuous gradient was generated to resolve the analytes by mixing solvent A (0.2% formic acid in water) with solvent B (0.2% formic acid in methanol) at different ratios starting from 0% B for 3 minutes, then linearly to 100% B over 3.5 min, then hold for 3 min, and then back to 0% B. [.sup.13C.sub.9, .sup.15N.sub.1]-tyrosine was included to simultaneously monitor for potential artificial generation of analyte. 3-bromotyrosine, tyrosine and their respective internal standard [.sup.13C.sub.6]-3-bromotyrosine and [.sup.13C.sub.9, .sup.15N.sub.1]-tyrosine and the artificial product, [.sup.13C.sub.9, .sup.15N.sub.1]-bromotyrosine were monitored using electrospray ionization in positive-ion mode with multiple reaction monitoring (MRM) of precursor and characteristic product-ion transitions of m/z 260.fwdarw.135, 182.fwdarw.136, 266.fwdarw.141, 192.fwdarw.145 and 270.fwdarw.144, amu, respectively. The parameters for the ion monitoring were optimized automatically. Nitrogen (99.95% purity) was used as the source and helium was used as collision gas. Various concentrations of nonisotopically labeled 3-bromotyrosine and tyrosine standard were spiked into control urine to prepare the calibration curves for quantification of 3-bromotyrosine and tyrosine, respectively. The internal standard [.sup.13C.sub.6]-3-bromotyrosine was used for quantification as well as to calculate recovery rate of 3-bromotyrosine (which was >80% based on separate control studies). Under the conditions employed for the assay, no artificial bromination was detected. Results were expressed as urine 3-bromotyrosine (ng/mg creatinine).

(31) For Free BrTyr+Glucuronidated BrTyr:

(32) 200 ul urine was aliquoted into a glass tube with 20 ul 0.5 uM [.sup.13C.sub.6]-BrTyr and 1 mM [.sup.13C.sub.9, .sup.15N.sub.1]-tyrosine added as internal standard. 100 ul 1 M ammonium acetate buffer, pH 5.0, was added to the urine sample followed by addition of 10 ul 16 mg/ml glucuronidase (G0751, Sigma) in water. The sample was kept at 45 C for 15 hours and then cooled down. The urine was diluted with 2 ml 0.1% formic acid in water and follow the same procedure as above for free BrTyr.

(33) 3-Bromotyroine Detection in Patients

(34) BrTyr tackles this unmet clinical need. BrTyr was measured at baseline (BL), and after 3 months of injections in 68 individuals with severe asthma. There is a wide range of BrTyr levels in asthmatics as at baseline (FIG. 1, Table 1). Free BrY is the naturally free bromotyrosine in a sample, and Total BrY is the total Free Bry+4-O-glucuronide-3-bromotyrosine. Total BrY is measured in this example by de-glurcuroniting the 4-O-glucuronide-3-bromotyrosine in a sample and then measuring the level of BrY present in the sample).

(35) TABLE-US-00003 TABLE 1 Baseline measurements of BrTyr Baseline (n = 68) Free BrY (ng/mg Cr) Median [25/75%] 0.487 [0.268-0.749] Mean [SE] 0.610 [0.062] Range [0%-100%] 0.074-2.729 Total BrY (ng/mg Cr) Median [25/75%] 13.64 [6.00-22.72] Mean [SE] 16.92 [1.78] Range [0%-100%]  1.94-77.16 % Free BrY [(Free/total) * 100] Median [25/75%]  3.68 [1.71-7.35] Mean [SE]  5.64 [0.7] Range [0%-100%]  0.87-33.07
Most of the BrTyr present in the total BrTyr is 4-O-glucurinided-3 bromotyrosine (% free BrTyr, median (IQR): 3.68 (1.71-7.35); Table 1). FIG. 2 demonstrates the correlation between baseline Total BrTyr and naturally Free BrTyr (R2=0.179, P=0.0003).

(36) All patients had uniform drop in eosinophils by 3 months (Bsln eosinophil numbers 440±77 vs. 3 months after mepolizumab 62±0.8; P<0.001), but overall BrTyr (free and total) levels did not significantly change among individuals with some dropping and others not (BrTyr (ng/mg Cr), mean±SE: Bsln Free BrTyr 0.610±0.0620 vs 3 months after mepolizumab 0.782±0.117, p=0.11; Bsln Total BrTyr 16.92±1.78 vs 3 months after mepolizumab 18.08±1.85, p=0.51).

(37) In this phenotype designated as Th2-high (by blood eosinophil counts), there existed a BrTyr subgroup of patients (45% of asthmatics have an decrease in BrTyr after mepolizumab) with activated eosinophils that were the most likely to respond to mepolizumab. These data also indicate that one in four asthmatics who have eosinophil counts>150, may have greater benefit from therapies targeting non-eosinophilic pathways (that are less expensive).

(38) The Asthma control test (ACT) is a numerical score to measure if asthma symptoms are well controlled. An increase of 3 points or more is considered likely evidence to indicate a clinically meaningful change in asthma control in an individual patient over time. More than 50% (53%) of the asthmatics showed an increase of 3 points or more in their ACT score after 3 months of mepolizumab. This also means that nearly half of the subjects on this highly expensive biologic agent failed to show benefit. Therefore, having a biomarker that can identify those who are more likely to benefit, and those less likely to benefit, is an important advance. The baseline BrTyr levels of Free as well as Total BrTyr, were significantly higher in the groups of asthmatics that showed a significant clinically relevant improvement (> or =3 point increase) in their asthma control test (FIG. 3).

(39) BrTyr as Predictor of Clinical Response to Mepolizumab

(40) Median, Tertiles, Quartiles and Receiver operating characteristic curve analyses were used to assess the utility of BrTyr measurements at baseline for the prediction of clinical responsiveness to mepolizumab. The clinical responsiveness in this Example was defined as an improvement of 3 or more point in their asthma control test after 3 months of mepolizumab. FIGS. 4 and 5 shows the ODD ratios for Free and total BrTyr as a predictor for mepolizumab response based on the asthma control test. Those with higher baseline BrTyr levels (both free and total) have increased likelihood of improvement in asthma questionnaire. Total BrTyr levels (measured after urine incubation with the glucuronidase enzyme digestion treatment) are 10-20 fold higher, and easier to measure, and show improved accuracy as well.

(41) There is a wide range of changes in BrTyr levels after 3 months of mepolizumab, as shown in Table 2.

(42) TABLE-US-00004 TABLE 2 The distribution of the delta free and Total BrTyr (3 months mepolizumab minus Baseline) (n = 68) Delta Free BrY (ng/mg Cr) Median [25/75%] 0.028 [−0.195-2.89] Mean [SE] 0.172 [0.122] Range [0-100%] −1.67-4.65 Total BrY (ng/mg Cr) Median [25/75%] 0.979 [−4.85-5.27] Mean [SE] 1.165 [1.77] Range [0-100%] −55.0-46.6
Both free and total BrTyr levels show large variation before vs 3 months following initiation of mepolizumab therapy. While not shown in Table 2, patients also show large variation in response to therapy (e.g., a large fraction fail to show benefit). This raised the possibility that those which showed the highest initial levels of eosinoiphilic tissue injury at baseline (highest free and/or Total Br Tyr level) or largest reduction in BrTyr level, are most likely to show benefit with therapy. The change in free and total BrTyr (BrTyr levels at 3 months mepolizumab minus baseline measurement of BrTyr) is not associated with the change in asthma control test.
Steroids Decrease BrTyr

(43) As a site in the ongoing longitudinal NHLBI SARP3, we are evaluating asthmatics (severe and non-severe) that are stable on their current medications at baseline enrollment and then 2-3 weeks after intramuscular triamcinolone injection. BrTyr was measured at baseline (BL), and 2-3 weeks after intramuscular triamcinolone injection in 464 adults with asthma. There is a wide range of BrTyr levels in asthmatics at baseline (FIG. 6, Table 3). Most of the BrTyr present in the total measurement of BrTyr is present as 4-O-glucurinided-3 bromotyrosine (Table 3).

(44) TABLE-US-00005 TABLE 3 Baseline and 2-3 weeks after intramuscular triamcinolone injection measurements of BrTyr in Severe Asthma Research Cohort After 2-3 wks of intramuscular Baseline triamcinolone (n = 464) injection (n = 464) Free BrY (ng/mg Cr) Median [25/75%] 0.139 [0.063-0.25] 0.112 [0.061-0.224] Mean [SE] 0.241 [0.017] 0.208 [0.015] Range {0-100%] 0.003-4.149 0.002-3.720 Total BrY (ng/mg Cr) Median [25/75%]  2.28 [1.27-4.01]  1.93 [1.01-3.43]* Mean [SE]  3.50 [0.23]  2.93 [0.17] Range {0-100%]  0.23-74  0.11-46.4 % Free BrY [(Free/total) * 100] Median [25/75%]  5.89 [2.41-13.86]  6.96 [2.88-13.94] Mean [SE] 10.91 [0.62] 11.51 [0.62] Range [0%-100%]  0.04 89.6  0.18-88.1]
FIG. 6 demonstrate the correlation between baseline Total BrTyr and Free BrTyr (R2=0.029, P=0.0002), showing minimal association (measurement of total BrTyr only explains 2.9% of the variation in free BrTyr). Interestingly, Total BrTyr decreases significantly after intramuscular triamcinolone injection (p=0.018) whereas no significant change is found with Free BrTyr (p=0.08).

(45) In summary, the above work asked whether free or Total BrTyr predicts likelihood of exacerbation during a steroid withdraw study in mild asthmatics. Subjects were given an intramuscular injection of steroid, and then the frequency of worsening of asthma symptoms monitored over time during the steroid withdraw period. Again note that Total BrTyr is nearly 50-fold higher than free BrTyr, indicating the vast majority of BrTyr is glucuroinidated and not monitored when quantifying free BrTyr in urine without the glucuronidase enzyme digestion step. Also note in FIG. 7 that Free vs Total BrTyr are poorly correlated. Only 2.9% of the variation in Free BrTyr levels is explained by variations in Total BrTyr levels (they are essentially independent values).

(46) Asthmatics with unscheduled doctor (MD) visits and emergency room (ER) visits in the last 12 months had significantly higher total BrTyr levels at baseline whereas no difference was found with Free BrTyr (Table 4, FIG. 7).

(47) TABLE-US-00006 TABLE 4 Comparison of asthma phenotypes and Baseline measurements of BrTyr in Severe Asthma Research Cohort Free Phenotype (N) BrTyr Total Brtyr SARP3 Nonsevere (204) 0.23 (0.03) 3.27 (0.23) SARP3 Severe (317) 0.25 (0.38) 3.71 (0.33) P 0.5 0.3 No unscheduled MD visits last year (276) 0.23 (0.02) 3.13 (0.21) Yes, Unscheduled MD visits last year (213) 0.26 (0.03) 4.05 (0.43) P 0.4 0.0141 No ED visits (373) 0.24 (0.02) 3.26 (0.18) Yes, ED visit last year (116) 0.25 (0.04) 4.43 (0.72) P 0.7 0.042
Data are represented as mean and SD. P-values are based on nonparametric t-test It is clear that total BrTyr levels predict likelihood of unscheduled MD visit or ED visit.

(48) Median and Tertiles were used to assess the utility of BrTyr measurements at baseline for the prediction of asthma control based on unscheduled doctor visits. High levels of Total BrTyr predict an increased risk for unscheduled doctor visits, whereas no correlation was found with free BrTyr. (FIG. 8) FIG. 9 shows ORs and 95% CI for the associations between Total BrTyr levels and unscheduled MD visits. Results shown represent the ORs (circles) and 95% CI (lines) of unscheduled MD visited and (A) high level of bromotyrosine (greater than median level of 2.25 ng/mg cr, filled circle) versus not a high level (open circle) and (B) high level of bromotyrosine (greater than <1.60 ng/mg Cr), filled circle) versus not a high level (open circle). Subjects with higher level of urinary total BrTyr were more likely to experience unscheduled MD visits. When Total urinary BrTyr is measured, whether one stratifies above v below median value, or by tertile levels, one sees higher levels of BrTyr predict worse outcome, and more unscheduled MD visits for worsening asthma symptoms. Thus, Total BrTyr dose dependently predicts worse outcome.

REFERENCES

(49) 1. J. Pediatrics, 2011, 159, 248-255—Samuel H. Wedges et. al. 2. Clin. Trans. Science, 2, 2009, 112-117—Samuel H Wedges et. al 3. J. Allergy Clin Immunol, 2015, 135 (4) 877-883—Douglas C. Cowan et. al. 4. U.S. Pat. No. 6,306,576—Diagnostic methods for asthma

(50) All publications and patents mentioned in the present application are herein incorporated by reference. Various modification and variation of the described methods and compositions of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the relevant fields are intended to be within the scope of the following claims.