WO2014059197A1

WO2014059197A1 - Methods and compositions for diagnosing and treating hyposmia

Info

Publication number: WO2014059197A1
Application number: PCT/US2013/064416
Authority: WO
Inventors: Robert I. Henkin
Original assignee: Henkin Robert I
Priority date: 2012-10-10
Filing date: 2013-10-10
Publication date: 2014-04-17

Abstract

Disclosed herein are methods for diagnosing a subject with a taste or smell disorder, said methods comprising measuring a level of one or more biomarkers present in a biological sample, wherein said biomarkers are selected from the group consisting of IL-1, IL- 1α, IL-Ιβ, IL-1Ra, IL-1RII, IL-2, IL-2R, IL-6, IL-10, IL18, IFN-β, IFN-γ, IgE and eosinophils. Also disclosed herein are methods and compositions for treating a subject for a taste or smell disorder.

Description

METHODS AND COMPOSITIONS FOR DIAGNOSING AND TREATING HYPOSMIA

CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application Nos. 61/712,088, filed October 10, 2012; 61/717,452, filed October 23, 2012; 61/772,520, filed March 4, 2013;

61/802,314, filed March 15, 2013; 61/814,187, filed April 19, 2013; and 61/838,748, filed June 24, 2013; each of which application is incorporated herein by reference in its entirety.

BACKGROUND OF THE DISCLOSURE

[0002] Hyposmia is a reduced ability to smell and detect odors. Anosmia is a complete loss of ability to smell and detect odors. There is a need in the art for methods for diagnosing and treating smell loss.

INCORPORATION BY REFERENCE

[0003] All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. In the event of a conflict between a term herein and a term incorporated by reference, the term herein controls.

SUMMARY OF THE DISCLOSURE

[0004] Disclosed herein are methods of diagnosing a taste or smell disorder in a subject.

[0005] In one aspect, disclosed herein are methods of diagnosing a taste or smell disorder in a subject, the method comprising: (a) obtaining a biological sample from the subject; (b) measuring a level of one or more biomarkers present in the biological sample, wherein the one or more biomarkers are selected from the group consisting of IL-la, IL-Ιβ, IL-lra, IL-1 RII, IL-2, IL-2R, IL-6, IL-10, IL-18, IFN-β, IFN-γ, IgE, eosinophils, and any combination thereof; and (c) diagnosing the subject with the taste or smell disorder when the level(s) of the one or more biomarkers are abnormal. Some embodiments further comprise treating the subject diagnosed with the taste or smell disorder. In some embodiments, the diagnosing in (c) is computer implemented. Some embodiments further comprise sending a result from the diagnosing to a party via a communication medium.

[0006] In some embodiments, the diagnosing comprises determining that the level of the one or more biomarkers, individually, is one selected from the group consisting of: above a threshold level, below a threshold level, and within a range that is indicative of having taste or smell disorders. In some embodiments, the threshold level is an average level as measured in a control population. In some embodiments, the threshold level is at least 1.5 times higher or lower than an average level as measured in a control population.

[0007] In some embodiments, the biological sample comprises a blood sample, a plasma sample, a urine sample, a saliva sample, a nasal mucus sample, or a combination thereof.

[0008] In some embodiments, the measuring comprises measuring an eosinophil level, and the diagnosing comprises determining that the eosinophil level is above a threshold level. In some embodiments, the biological sample comprises a blood sample or a plasma sample, and wherein the threshold level is from 300 cells/HPF (high powered field) to 400 cells/HPF. In some embodiments, the biological sample comprises a blood sample or a plasma sample, and wherein the threshold level is about: 300 cells/HPF, 350 cells/HPF, or 400 cells/HPF. In some

embodiments, measuring the eosiniphil level is performed with a Coulter counter.

[0009] In some embodiments, the measuring comprises measuring an IgE level, and the diagnosing comprises determining that the IgE level is above a threshold level. In some embodiments, the biological sample comprises a blood sample or a plasma sample, and wherein the threshold level is from 75 kU/L to 125 kU/L. In some embodiments, the biological sample comprises a blood sample or a plasma sample, and wherein the threshold level is about: 75 kU/L, 100 kU/L, or 125 kU/L. In some embodiments, measuring the IgE level comprises a fluorescence polarization assay.

[0010] In some embodiments, the measuring comprises measuring a level of one or more biomarkers selected from the group consisting of IL-la, IL-Ιβ, IL-6, IL-18, and any combination thereof, and the diagnosing comprises determining that level(s) are above a threshold level.

Some embodiments comprise measuring the IL-6 level. In some embodiments, the biological sample is a nasal mucus sample, and wherein the threshold level is from 5 pg/mL to about 15 pg/mL. In some embodiments, the biological sample is a plasma sample, and wherein the threshold level is from 0.05 pg/mL to about 0.2 pg/mL. In some embodiments, the biological sample is a saliva sample, and wherein the threshold level is from 0.15 pg/mL to about 0.4 pg/mL.

[0011] In some embodiments, the measuring comprises measuring a level of one or more biomarkers selected from the group consisting of IL-lra, IL-10, IFN-γ, and any combination thereof, and the diagnosing comprises determining that level(s) are below a threshold level.

[0012] Some embodiments comprise diagnosing the subject with the taste or smell disorder based upon one or more measurements comprising: (i) the level of IL-l that is about: 125 pg/mL to 195 pg/mL, 150 pg/mL to 170 pg/mL, 120 pg/mL to 170 pg/mL, or 150 pg/mL to 195 pg/mL; (ii) the level of IL-Ιβ that is about: 195 pg/mL to 300 pg/mL, 220 pg/mL to 275 pg/mL, 220 pg/mL to 300 pg/mL, or 195 pg/mL to 275 pg/mL; (iii) the level of IL-lra that is about: 30,000 pg/mL to 90,000 pg/mL, 45,000 pg/mL to 75,000 pg/mL, 45,000 pg/mL to 90,000 pg/mL, or 30,000 pg/mL to 75,000 pg/mL; (iv) the level of IL-1 RII that is about: 960 pg/mL to 2600 pg/mL, 1370 pg/mL to 2190 pg/mL, 1370 pg/mL to 2600 pg/mL, or 960 pg/mL to 2190 pg/mL; (v) the level of IL-2 that is about: 0 pg/mL, 0.1 pg/mL, 0.2 pg/mL, 0.3 pg/mL, or 0.5 pg/mL; (vi) the level of IL-2R that is about: 0 to 200 pg/mL, 50 to 150 pg/mL, 0 to 150 pg/mL, or 50 to 200 pg/mL; (vii) the level of IL-6 that is about: 0.1 pg/mL to 2.2 pg/mL, 0.6 pg/mL to 1.7 pg/mL, 0.6 pg/mL to 2.2 pg/mL, or 0.1 pg/mL to 1.7 pg/mL; (viii) the level of IL-10 that is about: 0 pg/mL to 3.5 pg/mL, 0.8 pg/mL to 2.7 pg/mL, 0.8 pg/mL to 3.5 pg/mL, or 0 pg/mL to 2.7 pg/mL; (ix) the level of IL-18 that is about: 40 pg/mL to 290 pg/mL, 100 pg/mL to 230 pg/mL, 40 pg/mL to 230 pg/mL, or 100 pg/mL to 290 pg/mL; (x) the level of IFN-β that is about: 0 pg/mL to 910 pg/mL, 230 pg/mL to 680 pg/mL, 230 pg/mL to 910 pg/mL, or 0 pg/mL to 680 pg/mL; or (xi) the level of IFN-γ that is about: 55 pg/mL to 110 pg/mL, 70 pg/mL to 95 pg/mL, 70 pg/mL to 110 pg/mL, 55 pg/mL to 95 pg/mL. In some embodiments, diagnosing is based upon 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 of the one or more measurements.

[0013] In some embodiments, measuring comprises using one or more techniques that are fluorescence microscopy, a radioimmunoassay, a fluorescence immunoassay, fluorescence- activated cell sorting, mass spectrometry, liquid chromatography, electrophoresis, protein arrays, or a combination thereof.

[0014] In some embodiments, measuring comprises using one or more antibodies that bind the one or more proteins. In some embodiments, at least one antibody is conjugated to an enzyme, a fluorescent molecule, or a radio-label. In some embodiments, the one or more antibodies are used in an immunostain, an immunoprecipitation, an Immunoelectrophoresis, an immunoblot, a western blot, a proximity ligation assay, or a spectrophotometry assay. In some embodiments, the one or more antibodies are used in the spectrophotometry assay that is an EMIT (Enzyme Multiplied Immunoassay Technique) assay, an ELISA (Enzyme Linked Immunosorbent Assay), a sandwich ELISA, or a competitive ELISA.

[0015] In some embodiments, the taste or smell disorder is anosmia, hyposmia, phantosmia, parosmia, ageusia, hypogeusia, phantageusia, or parageusia. In some embodiments, the taste or smell disorder is anosmia or hyposmia.

[0016] In some embodiments, treating comprises administering a pharmaceutical composition comprising an effective amount of one or more phosphodiesterase inhibitors to the subject. In some embodiments, pharmaceutical composition is administered orally. In some embodiments, pharmaceutical composition is administered intranasally. In some embodiments, the one or more PDE inhibitors comprise a non-selective PDE inhibitor, a PDE-1 selective inhibitor, a PDE-2 selective inhibitor, a PDE-3 selective inhibitor, a PDE-4 selective inhibitor, a PDE-5 selective inhibitor, or a combination thereof.

[0017] Also disclosed are methods of treating taste or smell disorders in a subject in need thereof, the methods comprising administering an effective amount of an inhibitor of a proinflammatory cytokine. In some embodiments, the inhibitor is an antibody, an antibody fragment, or an antibody mimetic. In some embodiments, the antibody, antibody fragment, or antibody mimetic binds to the pro -inflammatory cytokine. In some embodiments, the antibody, antibody fragment, or antibody mimetic binds to a receptor for the pro -inflammatory cytokine. In some embodiments, the pro-inflammatory cytokine is IL-6 and the inhibitor is tociluzumab, sarilumab, elsilimomab, siltuximab, sirukumab, BMS-945429, CDP6038, VX30, ARGX-109, FM101, or lunasin. In some embodiments, the pro-inflammatory cytokine is IL-l and the inhibitor is IL-1RA. In some embodiments, the pro-inflammatory cytokine IL-Ιβ and the inhibitor is canakinumab. In some embodiments, the pro -inflammatory cytokine is TNF-a and the inhibitor is infliximab; adalimumab; certolizumab pegol; golimumab; etanercept; a xanthine derivative that is pentoxifylline; bupropion; or a 5-HT2A agonist that is (R)-DOI (2,5- dimethoxy-4-iodoamphetamine), TCB-2 (l-[(7R)-3-bromo-2,5-dimethoxybicyclo[4.2.0]octa- l,3,5-trien-7-yl]methanamine), LSD (lysergic acid diethylamide), or LSZ (Lysergic acid 2,4- dimethy lazetidide) .

[0018] Also disclosed are methods of treating a taste or smell disorder in a subject in need thereof, the methods comprising administering a pharmaceutical composition an effective amount of an adenylyl cyclase activator, a guanylyl cyclase activator, a cAMP analog, a cGMP analog, or a combination thereof. In some embodiments, the pharmaceutical composition comprises the adenylyl cyclase activator that is forskolin; 1,9-Dideoxyforskolin; 6-[3- (dimethylamino)propionyl]forskolin; adenylyl cyclase toxin; NB001 ; NKH 477; Pituitary adenylate cyclase activating polypeptide-38; Pituitary adenylate cyclase activating polypeptide- 27; or a combination thereof. In some embodiments, the pharmaceutical composition comprises the guanylyl cyclase activator that is A-50619 hydrochloride; atriopeptin II; 6P-Hydroxy-8,13- epoxy-labd-14-en-l 1-one; 9a-Hydroxy-8,13-epoxy-labd-14-en-l 1-one; isoliquiritigenin;

protoporphyrin IX; YC-1; BAY41-2272; CMF-1571; A-350619; BAY 41-8543; BAY 63-2521; BAY58-2667; HMR1766; S3448; or a combination thereof.

[0019] Also disclosed are methods of treating a taste or smell disorder in a subject in need thereof, the method comprising administering a pharmaceutical composition comprising an effective amount of an anti-inflammatory cytokine to the subject. In some embodiments, the one or more anti- inflammatory cytokines comprise IL-lra, IL-10, IFN-γ, or a combination thereof.

[0020] In some embodiments, the pharmaceutical composition is administered one, two, three, or more times per day. In some embodiments, the pharmaceutical composition is administered each day for from 7 days to 5 years, 7 days to 1 year, 7 days to 6 months, 7 days to 3 months, or 7 days to 1 month. In some embodiments, the pharmaceutical composition is administered each day on a continuous basis.

[0021] In some embodiments, the subject in need thereof experiences a decrease in a detection threshold (DT) score or a recognition threshold (RT) score for at least one tastant or odorant following administration of the pharmaceutical composition. In some embodiments, the subject experiences an increase in a magnitude estimation (ME) score for at least one tastant or odorant following administration of the pharmaceutical composition. In some embodiments, the subject in need thereof experiences a decrease in phantosmia, parosmia, phantageusia, or parageusia following administration of the pharmaceutical composition. In some embodiments, a proinflammatory cytokine level in the subject is lower following administration of the

pharmaceutical composition. In some embodiments, eosinophil levels in the subject are lower following administration of the pharmaceutical composition. In some embodiments, IgE levels in the subject are lower following administration of the pharmaceutical composition. In some embodiments, a cyclic nucleotide level in the subject is higher following administration of the pharmaceutical composition.

[0022] In some embodiments, the pharmaceutical composition is administered orally. In some embodiments, the pharmaceutical composition is administered intranasally.

[0023] In some embodiments, the subject in need thereof was diagnosed with a taste or smell disorder according to any of the diagnostic methods disclosed herein.

[0024]

[0025] In some methods of diagnosing a taste or smell disorder in a subject, the methods comprise: (a) measuring a level of one or more pro-inflammatory cytokines selected from the group consisting of IL-la, IL-Ιβ, IL-6, IL-18 in a biological sample from the subject; and (b) diagnosing the subject with the taste or smell disorder based upon the level of one or more proinflammatory cytokines that is higher than a threshold level.

[0026] In some embodiments, the threshold level is an average level of the one or more proinflammatory cytokines as measured in a control population. In some embodiments, the threshold level is at least 1.5 times an average level of the one or more pro-inflammatory cytokines as measured in a control population. [0027] In some embodiments, the biological sample comprises a blood sample, a plasma sample, a urine sample, a saliva sample, a nasal mucus sample, or a combination thereof. In some embodiments, the biological sample comprises a plasma sample, a urine sample, a saliva sample, or a nasal mucus sample.

[0028] In some embodiments, measuring comprises using one or more antibodies that bind the one or more pro-inflammatory cytokines. In some embodiments, at least one antibody is conjugated to an enzyme, a fluorescent molecule, or a radio-label. In some embodiments, the one or more antibodies are used in an immunostain, an immunoprecipitation, an

Immunoelectrophoresis, an immunoblot, a western blot, a proximity ligation assay, or a spectrophotometry assay. In some embodiments, the one or more antibodies are used in the spectrophotometry assay that is an EMIT (Enzyme Multiplied Immunoassay Technique) assay, an ELISA (Enzyme Linked Immunosorbent Assay), a sandwich ELISA, or a competitive ELISA.

[0029] Some embodiments further comprise measuring a level of one or more anti-inflammatory cytokines in the biological samples. In some embodiments, the one or more anti-inflammatory cytokines comprise IL-lra, IL-10, IFN-γ, or a combination thereof. In some embodiments, diagnosing is further based upon the level of at least one anti-inflammatory cytokine being lower than a second threshold level. In some embodiments, the second threshold level is an average anti-inflammatory cytokine level as measured in a control population. In some embodiments, the second threshold level is at least 1.5 times lower than an average anti- inflammatory cytokine level as measured in a control population.

[0030] In some methods of diagnosing a taste or smell disorder in a subject, the methods comprise: (a) measuring a level of IL-6 in a biological sample from the subject; (b) diagnosing the subject with the taste or smell disorder based upon the level of IL-6 that is higher than a threshold level.

[0031] In some embodiments, the threshold level is an average level of the IL-6 as measured in a control population. In some embodiments, the threshold level is at least 1.5 times an average level of the IL-6 as measured in a control population.

[0032] In some embodiments, the biological sample comprises a blood sample, a plasma sample, a urine sample, a saliva sample, a nasal mucus sample, or a combination thereof. In some embodiments, the biological sample comprises a plasma sample, a urine sample, a saliva sample, or a nasal mucus sample.

In some embodiments, the biological sample is a nasal mucus sample. In some embodiments, the threshold level is from 5 pg/mL to about 15 pg/mL. [0033] In some embodiments, the biological sample is a plasma sample. In some embodiments, the threshold level is from 0.05 pg/mL to about 0.2 pg/mL.

[0034] In some embodiments, the biological sample is a saliva sample. In some embodiments, the threshold level is from 0.15 pg/mL to about 0.4 pg/mL.

[0035] In some embodiments, measuring comprises using an antibody that binds IL-6. In some embodiments, the antibody is conjugated to an enzyme, a fluorescent molecule, or a radio-label. In some embodiments, the antibody is used in an immunostain, an immunoprecipitation, an Immunoelectrophoresis, an immunoblot, a western blot, a proximity ligation assay, or a spectrophotometry assay. In some embodiments, the antibody is used in the spectrophotometry assay that is an EMIT (Enzyme Multiplied Immunoassay Technique) assay, an ELISA (Enzyme Linked Immunosorbent Assay), a sandwich ELISA, or a competitive ELISA.

[0036] In some methods of diagnosing a taste or smell disorder in a subject, the methods comprise: (a) measuring a level of IgE protein in a biological sample from the subject; and (b) diagnosing the subject with the taste or smell disorder based upon the level of IgE protein that is higher than a threshold level.

[0037] In some embodiments, the threshold level is an average level of IgE protein as measured in a control population. In some embodiments, the threshold level is at least 1.5 times an average level of IgE protein as measured in a control population.

[0038] In some embodiments, the biological sample comprises a blood sample, a plasma sample, a urine sample, a saliva sample, a nasal mucus sample, or a combination thereof. In some embodiments, the biological sample comprises a blood sample or a plasma sample. In some embodiments, the threshold level is from 75 kU/L to 125 kU/L. In some embodiments, the threshold level is about: 75 kU/L, 100 kU/L, or 125 kU/L. In some embodiments, measuring the level of IgE protein comprises a fluorescence polarization assay.

[0039] In some methods of diagnosing a taste or smell disorder in a subject, the methods comprise: (a) measuring a level of eosiniphils in a biological sample from the subject; and (b) diagnosing the subject with the taste or smell disorder based upon the level of eosinophils that is higher than a threshold level.

[0040] In some embodiments, the threshold level is an average level of eosinophils as measured in a control population. In some embodiments, the threshold level is at least 1.5 times an average level of eosinophils as measured in a control population.

[0041] In some embodiments, the biological sample comprises a blood sample, a plasma sample, a urine sample, a saliva sample, a nasal mucus sample, or a combination thereof. In some embodiments, the biological sample comprises a blood sample or a plasma sample. In some embodiments, the threshold level is from 300 cells/HPF (high powered field) to 400 cells/HPF. In some embodiments, the threshold level is about: 300 cells/HPF, 350 cells/HPF, or 400 cells/HPF. In some embodiments, measuring the level of eosiniphils is performed with a Coulter counter.

[0042] In some methods of diagnosing a taste or smell disorder in a subject, the methods comprise: (a) measuring a level of one or more proteins selected from the group consisting of IL- la, IL-Ιβ, IL-lra, IL-1 RII, IL-2, IL-2R, IL-6, IL-10, IL-18, IFN-β, and IFN-γ in a nasal mucus sample from the subject; and (b) diagnosing the subject with the taste or smell disorder based upon one or more measurements comprising: (i) the level of IL-1 a that is about: 125 pg/mL to 195 pg/mL, 150 pg/mL to 170 pg/mL, 120 pg/mL to 170 pg/mL, or 150 pg/mL to 195 pg/mL; (ii) the level of IL-Ιβ that is about: 195 pg/mL to 300 pg/mL, 220 pg/mL to 275 pg/mL, 220 pg/mL to 300 pg/mL, or 195 pg/mL to 275 pg/mL; (iii) the level of IL-lra that is about: 30,000 pg/mL to 90,000 pg/mL, 45,000 pg/mL to 75,000 pg/mL, 45,000 pg/mL to 90,000 pg/mL, or 30,000 pg/mL to 75,000 pg/mL; (iv) the level of IL-1 RII that is about: 960 pg/mL to 2600 pg/mL, 1370 pg/mL to 2190 pg/mL, 1370 pg/mL to 2600 pg/mL, or 960 pg/mL to 2190 pg/mL; (v) the level of IL-2 that is about: 0 pg/mL, 0.1 pg/mL, 0.2 pg/mL, 0.3 pg/mL, or 0.5 pg/mL; (vi) the level of IL-2R that is about: 0 to 200 pg/mL, 50 to 150 pg/mL, 0 to 150 pg/mL, or 50 to 200 pg/mL; (vii) the level of IL-6 that is about: 0.1 pg/mL to 2.2 pg/mL, 0.6 pg/mL to 1.7 pg/mL, 0.6 pg/mL to 2.2 pg/mL, or 0.1 pg/mL to 1.7 pg/mL; (viii) the level of IL-10 that is about: 0 pg/mL to 3.5 pg/mL, 0.8 pg/mL to 2.7 pg/mL, 0.8 pg/mL to 3.5 pg/mL, or 0 pg/mL to 2.7 pg/mL; (ix) the level of IL-18 that is about: 40 pg/mL to 290 pg/mL, 100 pg/mL to 230 pg/mL, 40 pg/mL to 230 pg/mL, or 100 pg/mL to 290 pg/mL; (x) the level of IFN-β that is about: 0 pg/mL to 910 pg/mL, 230 pg/mL to 680 pg/mL, 230 pg/mL to 910 pg/mL, or 0 pg/mL to 680 pg/mL; or (xi) the level of IFN-γ that is about: 55 pg/mL to 110 pg/mL, 70 pg/mL to 95 pg/mL, 70 pg/mL to 110 pg/mL, 55 pg/mL to 95 pg/mL. In some embodiments, diagnosing is based upon 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 of the one or more measurements.

[0043] In some embodiments, measuring comprises using one or more antibodies that bind the one or more proteins. In some embodiments, at least one antibody is conjugated to an enzyme, a fluorescent molecule, or a radio-label. In some embodiments, the one or more antibodies are used in an immunostain, an immunoprecipitation, an Immunoelectrophoresis, an immunoblot, a western blot, a proximity ligation assay, or a spectrophotometry assay. In some embodiments, the one or more antibodies are used in the spectrophotometry assay that is an EMIT (Enzyme Multiplied Immunoassay Technique) assay, an ELISA (Enzyme Linked Immunosorbent Assay), a sandwich ELISA, or a competitive ELISA. [0044] In any of the diagnostic methods disclosed herein, the taste or smell disorder can be anosmia, hyposmia, phantosmia, parosmia, ageusia, hypogeusia, phantageusia, or parageusia. In some embodiments, the taste or smell disorder is anosmia or hyposmia.

[0045] Any of the diagnostic methods disclosed herein can further comprise measuring a level of one or more of cAMP, cGMP, or nitric oxide (NO) in a sample from the subject. In some embodiments, diagnosing is further based on the level of cAMP, cGMP, and/or nitric oxide (NO) that is lower than an average level measured in a control population.

[0046] In any of the diagnostic methods disclosed herein, measuring can comprise using one or more techniques that are fluorescence microscopy, a radioimmunoassay, a fluorescence immunoassay, fluorescence-activated cell sorting, mass spectrometry, liquid chromatography, electrophoresis, protein arrays, or a combination thereof.

[0047] In any of the diagnostic methods disclosed herein, the subject diagnosed with the taste or smell disorder can be further identified as a member of an orphan drug population.

[0048] Any of the diagnostic methods disclosed herein can further comprise evaluating the subject's taste or smell function by determining a detection threshold (DT) score, a recognition threshold (RT) score, a magnitude estimation (ME) score, a hedonic (H) score, or a combination thereof with a forced-choice, three-stimuli, stepwise-staircase technique using one or more tastants or odorants. In some embodiments, smell function is tested using the one or more odorants that comprise pyridine, nitrobenzene, thiophene, amyl acetate, or a combination thereof. In some embodiments, taste function is tested using the one or more tastants that comprise sodium chloride (NaCl), sucrose, hydrogen chloride (HC1), urea, or a combination thereof. In some embodiments, diagnosing is further based upon the DT score that is higher than an average DT score as measured in a control population. In some embodiments, diagnosing is further based upon the RT score that is higher than an average RT score as measured in a control population. In some embodiments, diagnosing is further based upon the ME score that is lower than an average ME score as measured in a control population. In some embodiments, diagnosing is further based upon the H score that is different than an average H score as measured in a control population.

[0049] Any of the diagnostic methods disclosed herein can further comprise treating the taste or smell disorder in the subject diagnosed with the taste or smell disorder. In some embodiments, the subject is a subject in need thereof.

[0050] In some embodiments, treating comprises administering a pharmaceutical composition to the subject. In some embodiments, the pharmaceutical composition is administered orally. In some embodiments, the pharmaceutical composition is administered intranasally. [0051] The pharmaceutical composition can comprise an effective amount of one or more phosphodiesterase inhibitors. In some embodiments, the one or more PDE inhibitors comprise a non-selective PDE inhibitor, a PDE-1 selective inhibitor, a PDE-2 selective inhibitor, a PDE-3 selective inhibitor, a PDE-4 selective inhibitor, a PDE-5 selective inhibitor, or a combination thereof. In some embodiments, the one or more PDE inhibitors comprise the non-selective PDE inhibitor that is a methylxanthine derivative. In some embodiments, the methylxanthine derivative is caffeine, theophylline, IBMX (3 -isobutyl-1 -methylxanthine) aminophylline, doxophylline, cipamphylline, neuphylline, pentoxiphylline, or diprophylline. In some

embodiments, the one or more PDE inhibitors comprise theophylline. Some embodiments comprise the PDE 1 inhibitor that is vinpocetine. Some embodiments comprise the PDE 2 inhibitor that is EHNA. Some embodiments comprise the PDE 3 inhibitor that is inamrinone, anagrelide, or cilostazol. Some embodiments comprise the PDE 4 inhibitor that is mesembrine, rolipram, ibudilast, piclamilast, luteolin, drotaverine, or roflumilast. Some embodiments comprise the PDE 5 inhibitor that is sildenafil, tadalafil, vardenafil, udenafil, avanafil, or dipyridamole. Some embodiments comprise the PDE 10 inhibitor that is papaverine. In some embodiments, the pharmaceutical composition is in a dosage unit that comprises less than 45 mg, 30 mg, 15 mg, 10 mg, 5 mg, 1 mg, 500 μg, 250 μg, 120 μg, 80 μg, 40 μg, or 20 μg individually of the one or more PDE inhibitors and a pharmaceutically acceptable carrier.

[0052] The pharmaceutical composition can comprise an effective amount of one or more antiinflammatory cytokines. In some embodiments, the one or more anti-inflammatory cytokines comprise IL-lra, IL-10, IFN-γ, or a combination thereof. In some embodiments, the

pharmaceutical composition comprises an effective amount of an antibody, antibody fragment, or antibody mimetic that inhibits a pro -inflammatory cytokine. In some embodiments, the antibody, antibody fragment, or antibody mimetic binds to the pro -inflammatory cytokines. In some embodiments, the antibody, antibody fragment, or antibody mimetic binds to a receptor the pro-inflammatory cytokines. In some embodiments, the pharmaceutical composition comprises the antibody that is a monoclonal antibody. In some embodiments, the monoclonal antibody is a recombinant antibody, a chimeric antibody, a human monoclonal antibody, or a humanized monoclonal antibody. In some embodiments, the pharmaceutical composition comprises the antibody fragment that is a FAB fragment, a FAB2 fragment, a Fv fragment, a ScFv fragment, an antibody light chain, or an antibody heavy chain. In some embodiments, the pharmaceutical composition comprises the antibody mimetic that is an affibody molecule, an affilin, an affitin, an anticalins, an avimers, a DARPins, a fynomer, a Kunitz domain peptide, or a monobody. [0053] The pharmaceutical composition can comprise an effective amount of an adenylyl cyclase activator, a guanylyl cyclase activator, a cAMP analog, a cGMP analog, or a

combination thereof. In some embodiments, the pharmaceutical composition comprises the adenylyl cyclase activator that is forskolin; 1,9-Dideoxyforskolin; 6-[3- (dimethylamino)propionyl]forskolin; adenylyl cyclase toxin; NB001 ; NKH 477; Pituitary adenylate cyclase activating polypeptide-38; Pituitary adenylate cyclase activating polypeptide- 27; or a combination thereof. In some embodiments, the pharmaceutical composition comprises the guanylyl cyclase activator that is A-50619 hydrochloride; atriopeptin II; 6P-Hydroxy-8,13- epoxy-labd-14-en-l 1-one; 9a-Hydroxy-8,13-epoxy-labd-14-en-l 1-one; isoliquiritigenin;

[0054] In some embodiments, the pharmaceutical composition further comprises a steroid. In some embodiments, the pharmaceutical composition does not comprise a steroid.

[0055] In some embodiments, the pharmaceutical composition further comprises a vasoactive agent that is a potassium channel activator, a calcium blocker, a beta-blocker, an alpha- adrenergic receptor antagonist, a dopamine agonist, an opioid antagonist, a prostaglandin, an endothelin antagonist, or a combination thereof.

[0056] Treatment efficacy can be demonstrated in a number of ways. In some embodiments, the subject experiences a decrease in a detection threshold (DT) score or a recognition threshold (RT) score as measured with a forced-choice, three-stimuli, stepwise-staircase technique using one or more tastants or odorants after treatment. In some embodiments, the subject experiences an increase in a magnitude estimation (ME) score as measured with a forced-choice, three- stimuli, stepwise-staircase technique using one or more tastants or odorants after treatment. In some embodiments, pro -inflammatory cytokine levels in the subject are lower after treatment. In some embodiments, eosinophil levels in the subject are lower after treatment. In some embodiments, IgE levels in the subject are lower after treatment.

[0057] Also disclosed herein are methods of treating taste or smell disorders in a subject in need thereof, the method comprising administering an effective amount of an inhibitor of a proinflammatory cytokine. In some embodiments, the inhibitor is an antibody, an antibody fragment, or an antibody mimetic. In some embodiments, the antibody, antibody fragment, or antibody mimetic binds to the pro -inflammatory cytokine. In some embodiments, the antibody, antibody fragment, or antibody mimetic binds to a receptor for the pro -inflammatory cytokine. In some embodiments, the pro-inflammatory cytokine is IL-l , IL-Ιβ, IL-6, IL-18, or TNF-a. In some embodiments, the inhibitor is administered intranasally. [0058] In some embodiments, the pro-inflammatory cytokine is IL-6. In some embodiments, the inhibitor is an antibody, antibody fragment, or antibody mimetic. In some embodiments, the antibody, antibody fragment, or antibody mimetic binds to IL-6. In some embodiments, the antibody, antibody fragment, or antibody mimetic binds to a receptor for IL-6. In some embodiments, the antibody, antibody fragment, or antibody mimetic is tociluzumab, sarilumab, elsilimomab, siltuximab, sirukumab, BMS-945429, CDP6038, VX30, ARGX-109, or FM101. In some embodiments, the inhibitor is lunasin.

[0059] In some embodiments, the pro-inflammatory cytokine is IL-l . In some embodiments, the inhibitor is an antibody, antibody fragment, or antibody mimetic. In some embodiments, the antibody, antibody fragment, or antibody mimetic binds to IL-l . In some embodiments, the antibody, antibody fragment, or antibody mimetic binds to a receptor for IL-l . In some embodiments, the inhibitor is IL-1RA.

[0060] In some embodiments, the pro-inflammatory cytokine is IL-Ιβ. In some embodiments, the inhibitor is an antibody, antibody fragment, or antibody mimetic. In some embodiments, the antibody, antibody fragment, or antibody mimetic binds to IL-Ιβ. In some embodiments, the antibody, antibody fragment, or antibody mimetic binds to a receptor for IL-Ιβ. In some embodiments, the antibody, antibody fragment, or antibody mimetic is canakinumab.

[0061] In some embodiments, the pro-inflammatory cytokine is TNF-a. In some embodiments, the inhibitor is an antibody, antibody fragment, or antibody mimetic. In some embodiments, the antibody, antibody fragment, or antibody mimetic binds to TNF-a. In some embodiments, the antibody, antibody fragment, or antibody mimetic binds to a receptor for TNF-a. In some embodiments, the antibody, antibody fragment, or antibody mimetic is infliximab, adalimumab, certolizumab pegol, or golimumab. In some embodiments, the inhibitor is etanercept, a xanthine derivative, bupropion, or a 5-HT2A agonist. In some embodiments, the inhibitor is the xanthine derivative that is pentoxifylline. In some embodiments, the inhibitor is the 5-HT2A agonist that is (R)-DOI (2,5-dimethoxy-4-iodoamphetamine), TCB-2 (l-[(7R)-3-bromo-2,5- dimethoxybicyclo[4.2.0]octa-l,3,5-trien-7-yl]methanamine), LSD (lysergic acid diethylamide), or LSZ (Lysergic acid 2,4-dimethylazetidide).

[0062] In some embodiments, the subject in need thereof experiences a decrease in a detection threshold (DT) score or a recognition threshold (RT) score for at least one tastant or odorant following administration of the inhibitor. In some embodiments, the subject experiences an increase in a magnitude estimation (ME) score for at least one tastant or odorant following administration of the inhibitor. In some embodiments, the subject in need thereof experiences a decrease in phantosmia, parosmia, phantageusia, or parageusia following administration of the inhibitor.

[0063] In another aspect, disclosed herein are methods of treating hyposmia or anosmia in a subject in need thereof, the methods comprising: transplanting a nasal mucus sample from a subject with normal olfactory function into the nasal cavity of the subject in need thereof, thereby treating hyposmia or anosmia. In some embodiments, the nasal mucus sample has a volume of about: 1-8 mL, 2-6 mL, 3-5 mL, 4 mL, 1 mL, 500 μί, 100 μί, or 50 μΐ,. Some embodiments further comprise performing a nasal lavage on the subject in need thereof prior to transplanting the nasal mucus sample.

[0064] Some embodiments further comprise evaluating the subject in need thereof s olfactory function by determining a detection threshold (DT) score, a recognition threshold (RT) score, a magnitude estimation (ME) score, or a combination thereof with a forced-choice, three-stimuli, stepwise-staircase technique using one or more olfaction testing compounds. In some

embodiments, the one or more olfaction testing compounds comprise pyridine, nitrobenzene, thiophene, amyl acetate, or a combination thereof. In some embodiments, the subject in need thereof s olfactory function is evaluated before and after transplanting the nasal mucus sample.

[0065] In some embodiments, (a) and (b) are repeated two or more times over a period of time. In some embodiments, the period of time is about: 20 years, 15 years, 10 years, 5 years, 1-365 days, 1-120 days, 1-90 days, 1-60 days, or 1-30 days. In some embodiments, (a) and (b) are repeated about: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more times.

[0066] Some embodiments further comprise at least partially sterilizing the nasal mucus sample prior to transplanting into the nasal cavity of the subject in need thereof. In some embodiments, the at least partially sterilizing comprises filtering the nasal mucus sample. In some

embodiments, the method does not comprise at least partially sterilizing the nasal mucus sample prior to transplanting into the nasal cavity of the subject in need thereof.

[0067] Some embodiments further comprise administering a steroid-free pharmaceutical dosage unit for intranasal administration comprising a positive amount of one or more PDE inhibitors effective for treating anosmia or hyposmia to the subject in need thereof. In some embodiments, the steroid- fee pharmaceutical dosage unit comprises the positive amount that is less than 45 mg, 30 mg, 15 mg, 10 mg, 5 mg, 1 mg, 500 μg, 250 μg, 120 μg, 80 μg, 40 μg, or 20 μg individually of the one or more PDE inhibitors and a pharmaceutically acceptable carrier.

[0068] In some embodiments, the one or more PDE inhibitors comprise a non-selective PDE inhibitor, a PDE-1 selective inhibitor, a PDE-2 selective inhibitor, a PDE-3 selective inhibitor, a PDE-4 selective inhibitor, a PDE-5 selective inhibitor, a PDE- 10 selective inhibitor, or a combination thereof. In some embodiments, the one or more PDE inhibitors comprise a nonselective PDE inhibitor that is a methylxanthine derivative. In some embodiments, the methylxanthine derivative is caffeine, theophylline, IBMX (3 -isobutyl-1 -methylxanthine) aminophylline, doxophylline, cipamphylline, neuphylline, pentoxiphylline, or diprophylline. In some embodiments, the methylxanthine derivative is theophylline.

[0069] Some embodiments comprise the PDE 1 inhibitor that is vinpocetine. Some embodiments comprise the PDE 2 inhibitor that is EHNA. Some embodiments comprise the PDE 3 inhibitor that is inamrinone, anagrelide, or cilostazol. Some embodiments comprise the PDE 4 inhibitor that is mesembrine, rolipram, ibudilast, piclamilast, luteolin, drotaverine, or roflumilast. Some embodiments comprise the PDE 5 inhibitor that is sildenafil, tadalafil, vardenafil, udenafil, avanafil, or dipyridamole. Some embodiments comprise the PDE 10 inhibitor that is papaverine.

[0070] In some embodiments, the steroid free pharmaceutical dosage unit is mixed with the nasal mucus sample prior to transplanting the nasal mucus sample into the nasal cavity of the subject in need thereof. In some embodiments, the steroid free pharmaceutical dosage unit is not mixed with the nasal mucus sample prior to transplanting the nasal mucus sample into the nasal cavity of the subject in need thereof.

[0071] In some embodiments, the subject in need thereof was diagnosed with hyposmia or anosmia according to any method disclosed herein.

[0072] Also disclosed herein are methods of treating hypogeusia or ageusia in a subject in need thereof, the methods comprising: transplanting a saliva sample from a subject with normal taste function into the oral cavity of the subject in need thereof, thereby treating hypogeusia or ageusia. In some embodiments, the saliva sample has a volume of about: 1-8 mL, 2-6 mL, 3-5 mL, 4 mL, 1 mL, 500 μί, 100 μί, or 50 μί.

[0073] Some embodiments further comprise evaluating the subject in need thereof s taste function by determining a detection threshold (DT) score, a recognition threshold (RT) score, a magnitude estimation (ME) score, or a combination thereof with a forced-choice, three-stimuli, stepwise-staircase technique using one or more tastants. In some embodiments, the one or more tastants comprise sodium chloride (NaCl), sucrose, hydrogen chloride (HC1), urea, or a combination thereof. In some embodiments, the subject in need thereof s taste function is evaluated before and after transplanting the saliva sample.

[0074] In some embodiments, (a) and (b) are repeated two or more times over a period of time. In some embodiments, the period of time is about: 20 years, 15 years, 10 years, 5 years, 1-365 days, 1-120 days, 1-90 days, 1-60 days, or 1-30 days. In some embodiments, (a) and (b) are repeated about: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more times.

[0075] Some embodiments further comprise at least partially sterilizing the saliva sample prior to transplanting into the oral cavity of the subject in need thereof. In some embodiments, the at least partially sterilizing comprises filtering the saliva sample. In some embodiments, the method does not comprise at least partially sterilizing the saliva sample prior to transplanting into the oral cavity of the subject in need thereof.

[0076] Some embodiments further comprise administering a steroid-free pharmaceutical dosage unit for intranasal administration comprising a positive amount of one or more PDE inhibitors effective for treating hypogeusia or ageusia to the subject in need thereof. In some embodiments, the steroid- fee pharmaceutical dosage unit comprises the positive amount that is less than 45 mg, 30 mg, 15 mg, 10 mg, 5 mg, 1 mg, 500 μg, 250 μg, 120 μg, 80 μg, 40 μg, or 20 μg individually of the one or more PDE inhibitors and a pharmaceutically acceptable carrier.

[0077] In some embodiments, the one or more PDE inhibitors comprise a non-selective PDE inhibitor, a PDE-1 selective inhibitor, a PDE-2 selective inhibitor, a PDE-3 selective inhibitor, a PDE-4 selective inhibitor, a PDE-5 selective inhibitor, a PDE- 10 selective inhibitor, or a combination thereof. In some embodiments, the one or more PDE inhibitors comprise a nonselective PDE inhibitor that is a methylxanthine derivative. In some embodiments, the methylxanthine derivative is caffeine, theophylline, IBMX (3 -isobutyl-1 -methylxanthine) aminophylline, doxophylline, cipamphylline, neuphylline, pentoxiphylline, or diprophylline. In some embodiments, the methylxanthine derivative is theophylline.

[0078] Some embodiments comprise the PDE 1 inhibitor that is vinpocetine. Some embodiments comprise the PDE 2 inhibitor that is EHNA. Some embodiments comprise the PDE 3 inhibitor that is inamrinone, anagrelide, or cilostazol. Some embodiments comprise the PDE 4 inhibitor that is mesembrine, rolipram, ibudilast, piclamilast, luteolin, drotaverine, or roflumilast. Some embodiments comprise the PDE 5 inhibitor that is sildenafil, tadalafil, vardenafil, udenafil, avanafil, or dipyridamole. Some embodiments comprise the PDE 10 inhibitor that is papaverine.

BRIEF DESCRIPTION OF THE DRAWINGS

[0079] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0080] Fig. 1: illustrates an exemplary courses of events related to a method of diagnosing a taste or smell disorder. [0081] Fig. 2: depicts a computer system useful for displaying, storing, retrieving, or calculating diagnostic results from a level of one or more biomarkers associated with taste or smell disorders; displaying, storing, retrieving, or calculating raw data from biomarker analysis; or displaying, storing, retrieving, or calculating any sample or subject information useful in the diagnostic methods disclosed herein.

DETAILED DESCRIPTION OF THE DISCLOSURE

[0082] The following description and examples illustrate embodiments of the invention in detail. It is to be understood that this invention is not limited to the particular embodiments described herein and as such can vary. Those of skill in the art will recognize that there are numerous variations and modifications of this invention, which are encompassed within its scope.

DEFINITIONS

[0083] Unless characterized differently, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0084] The term "about" in relation to a reference numerical value includes a range of values plus or minus 15% from that value. All numerical indications in this specification are to be understood as being qualified by the term "about," unless the context clearly indicates otherwise.

[0085] The term "or" can be used conjunctively or disjunctively.

[0086] The term "diagnosis" as used herein and its grammatical equivalents, means the testing of subjects to determine if they have a particular trait for use in a clinical decision. Diagnosis includes testing of subjects at risk of developing a particular disease resulting from infection by an infectious organism or a non infectious disease, such as cancer or a metabolic disease.

Diagnosis also includes testing of subjects who have developed particular symptoms to determine the cause of the symptoms. Diagnosis also includes prognosis, monitoring progress of a disease, and monitoring the efficacy of therapeutic regimens. The result of a diagnosis can be used to classify patients into groups for performance of clinical trials for administration of certain therapies.

[0087] The term "drug" as used herein, means any compounds of any degree of complexity that perturbs a biological state, whether by known or unknown mechanisms and whether or not they are used therapeutically. Drugs thus include: typical small molecules of research or therapeutic interest; naturally-occurring factors, such as endocrine, paracrine, or autocrine factors or factors interacting with cell receptors of all types; intracellular factors, such as elements of intracellular signaling pathways; factors isolated from other natural sources; pesticides; herbicides; and insecticides. [0088] "Phosphodiesterase inhibitor" or "PDE inhibitor" refers to any compound that inhibits a phosphodiesterase enzyme, isozyme or allozyme. The term refers to selective or non-selective inhibitors of cyclic guanosine 3',5'-monophosphate phosphodiesterases (cGMP-PDE) and cyclic adenosine 3',5'-monophosphate phosphodiesterases (cAMP-PDE).

[0089] The term "treating" and its grammatical equivalents as used herein include achieving a therapeutic benefit and/or a prophylactic benefit. By therapeutic benefit is meant eradication or amelioration of the underlying disorder being treated. Also, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the patient, notwithstanding that the patient may still be afflicted with the underlying disorder. For prophylactic benefit, the compositions may be administered to a patient at risk of developing a particular disease, or to a patient reporting one or more of the physiological symptoms of a disease, even though a diagnosis of this disease may not have been made.

[0090] "Therapeutically effective amount" refers to the amount of an active ingredient, with or without additional active ingredients, which is effective to achieve its intended purpose. While individual patient needs may vary, determination of optimal ranges for effective amounts of the compounds and compositions is within the skill of an ordinary practitioner of the art. Generally, the dosage required to provide an effective amount of the composition, and which can be adjusted by one of ordinary skill in the art, can vary depending on the age, health, physical condition, sex, weight, extent of the dysfunction of the recipient, frequency of treatment and the nature and scope of the dysfunction.

[0091] "Dosage unit" as used herein, refers to a discrete amount of a pharmaceutical

composition that is administered in a single event or package. The meaning of the term dosage unit is context specific. For example, a dosage unit for an intranasally administered liquid pharmaceutical composition would be the volume of the composition that is administered in a single event. For a nasal spray, the dosage unit would be the volume of the composition that is released upon each actuation of the nasal spray device. For a solid composition, a single dosage unit can, for example, be a single pill, tablet, or capsule.

[0092] "Patient" or "subject" includes mammals, such as humans, including those in need of treatment thereof. Humans can include, e.g., babies, children, teenagers, adults, and the elderly.

[0093] "Magnitude estimation" or "ME" is a measurement of the ability of a subject to determine the strength of a stimulant, such as an odorant or a tastant.

[0094] "Recognition threshold" or "RT" is a measurement of the ability of a subject to recognize the identity of a stimulant, such as an odorant or a tastant. [0095] "Detection threshold" or "DT" is a measurement of the ability of a subject to recognize exposure to a stimulant, such as an odorant or a tastant.

[0096] A "hedonic" value or "H" value is a measurement of a subject's reaction to a stimulant, such as an odorant or a tastant, as being pleasant or unpleasant.

[0097] "Hyposmia" is a smell disorder characterized by a reduced ability to smell and detect odors.

[0098] "Anosmia" is a smell disorder characterized by the complete loss of the ability to smell and detect odors.

[0099] "Phantosmia" is a smell disorder characterized by hallucination of smells, often without a stimulus. Often, the phantom odor is perceived as unpleasant.

[00100] "Parosmia" is is a smell disorder characterized by characterized by some odors being interpreted as other odors.

[00101] "Hypogeusia" is a taste disorder characterized by a reduced ability to taste.

[00102] "Ageusia" is a taste disorder characterized by a complete lack of the ability to taste.

[00103] "Phantageusia" is a taste disorder characterized by a hallucination of taste, often without a stimulus. Often the phantom taste is unpleasant.

[00104] "Parageusia" is a taste disorder characterized by characterized by some tastes being interpreted as other tastes.

METHODS OF THE INVENTION

[00105] Disclosed herein are methods for diagnosing a subject with taste or smell disorders. Taste or smell disorders can include, e.g., hyposmia, anosmia, phantosmia, parosmia, hypogeusia, ageusia, phantageusia, or parageusia. Also disclosed herein are methods and composition for treating a subject for a taste or smell disorder. The subject can have been diagnosed with the taste or smell disorder according to any of the methods disclosed herein. The methods of treatment can comprise administering a pharmaceutical composition. The methods of treatment can comprise transplanting a biological substance from a subject with normal taste and smell function to a subject with taste or smell loss.

[00106] The methods disclosed herein can comprise analyzing one or more biological samples from a subject to determine a level of one or more biological substances. The one or more biological samples can comprise, but are not limited to, a blood sample, a plasma sample, a urine sample, a saliva sample, a nasal mucus sample, or a combination thereof. In some embodiments, the one or more biological samples comprise the nasal mucus sample. Use of nasal specimens (e.g., the nasal mucus sample) provides a minimally invasive manner of obtaining biological samples for analysis. The results of this analysis are then suitable for use in diagnosis, prognosis, and determination of suitability of therapeutic interventions.

[00107] The term "biological substance" as used herein, and its grammatical equivalents, includes cells and their extra-cellular and intra-cellular constituents. For example, biological substances include pathogens, metabolites, DNA, R A, lipids, proteins, carbohydrates, receptors, enzymes, hormones, growth factors, growth inhibitory factors, cells, organs, tissues, portions of cells, tissues, or organs, subcellular organelles, chemically reactive molecules like H⁺, superoxides, ATP, citric acid, protein albumin, as well as combinations or aggregate representations of these types of biological variables. In addition, biological substances can include therapeutic agents such as methotrexate, steroids, non-steroidal anti-inflammatory drugs, soluble TNF-alpha receptor, TNF-alpha antibody, and interleukin-1 receptor antagonists.

[00108] Biological substances can comprise cytokines, such a pro-inflammatory cytokines or anti- inflammatory cytokines. Pro -inflammatory cytokines can include IL-l , IL-Ι β, IL-6, IL- 18, TNF-a, or a combination thereof. Anti- inflammatory cytokines can include IL-lra, IL-10, IFN-γ, or a combination thereof. The balance of pro- and anti-inflammatory cytokines can indicate whether a subject has a taste or smell disorder.

[00109] Biological substances can comprise cytokine receptors such as type I cytokine receptors, type II cytokine receptors, members of the immunoglobulin superfamily, members of the tumor necrosis factor receptor family, chemokine receptors, and or TGF beta receptors. In some embodiments, a cytokine receptor is IL-1 RII and/or IL-2R.

[00110] Biological substances can comprise eosinophils. Biological substances can comprise IgE protein. Biological substances can comprise cyclic nucleotides (e.g., cAMP, cGMP). Biological substances can comprise nitric oxide (NO).

[00111] The levels of the one or more biological substances can be compared,

individually, to a threshold level. Threshold levels, as described herein, can be an average level for a particular biological substance as measured in a control population, e.g., comprising subjects with normal taste and/or smell function. Threshold levels can be about: 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2 or more times higher or lower than an average level in a control population. In some embodiments, if the level of the biological substance is above or below the threshold level for the biological substance, the subject may be diagnosed with and/or treated for a taste or smell disorder. SAMPLE COLLECTION

[00112] One or more biological samples can be collected from a subject for analysis. In some embodiments, the one or more biological samples comprise a blood sample, a plasma sample, a urine sample, a saliva sample, a nasal mucus sample, or a combination thereof. In some embodiments, the one or more biological samples comprise the nasal mucus sample.

[00113] The one or more biological specimens can be from the nasal area (e.g., a nasal mucus sample). In some embodiments of the invention, a sample of nasal secretions is collected directly from the nose into a collection tube or device. In other embodiments of the invention, a sample of nasal secretion is collected on a sample collection device by passing it into the nostril of a patient. The device may be inserted sequentially into each nostril of the patient and advanced parallel to the hard palate with slow rotation. The device is then typically transferred to a transport tube, such as a glass or plastic test tube. The transport tube may include a suitable volume of a sterile medium such as ethanol or the like.

[00114] Suitable sample collection devices and methods are well known to those skilled in the art.

[00115] A saliva sample can be obtained by draining, spitting, suction, and/or swabbing.

Gustatory or masticatory stimulation can be used to increase the flow of saliva.

[00116] A blood sample can be collected, for example, by venipuncture, of finger sticking.

Blood samples can be collected, for example, in a tube (e.g., a vacuum tube, a capillary tube), a syringe, or a bag. Plasma samples can be derived from blood samples, e.g., by centrifugation.

[00117] A urine sample can be collected, e.g., in a cup.

[00118] A nasal sample collection device can be a swab, a wooden spatula, bibulous materials such as a cotton ball, filter, or gauze pad, an absorbent-tipped applicator, capillary tube, or a pipette. A swab can be used as a sample collection device, and the sample processing element comprises a swab holder or a swab processing insert. The swab holder or swab processing insert can be tapered or angled to allow a single sample processing element to accommodate all types of swabs by allowing swabs with different amounts of fiber, or that are wound to different levels of tightness, to be held securely within the holder or insert. Most preferably, the swab holder or swab processing insert securely holds the swab to provide stability.

[00119] In some instances, samples may be collected from individuals repeatedly over a longitudinal period of time (e.g., once a day, once a week, once a month, biannually or annually). Obtaining numerous samples from an individual over a period of time can be used to verify results from earlier detections and/or to identify an alteration as a result of, for example, drug treatment. Samples can be obtained from humans or non-humans.

ANALYSIS

[00120] One or more biological samples can be collected and analyzed using one or more analytical techniques including enzymatic technique, ELISA, fluorometric technique, mass spectrography, HPLC, GLC, PCR, and other similar techniques. The analysis can comprise determining the presence and/or level of one or more biological substance in the one or more biological sample.

Polymerase Chain Reaction (PCR)

[00121] The polymerase chain reaction (PCR) is a process for amplifying one or more desired specific nucleic acid sequences found in a nucleic acid. Because large amounts of a specific sequence may be produced by this process, it is used for improving the efficiency of cloning DNA or messenger RNA and for amplifying a target sequence to facilitate detection thereof.

[00122] PCR involves a chain reaction for producing, in exponential quantities relative to the number of reaction steps involved, at least one specific nucleic acid sequence given (a) that the ends of the required sequence are known in sufficient detail that oligonucleotides can be synthesized which will hybridize to them, and (b) that a small amount of the sequence is available to initiate the chain reaction. The product of the chain reaction would be a discrete nucleic acid duplex with termini corresponding to the ends of the specific primers employed.

[00123] Any source of nucleic acid, in purified or non purified form, can be utilized as the starting nucleic acid or acids, provided it contains or is suspected of containing the specific nucleic acid sequence desired. Thus, the process may employ, for example, DNA or RNA, including messenger RNA, which DNA or RNA may be single stranded or double stranded. In addition, a DNA-RNA hybrid which contains one strand of each may be utilized. A mixture of any of these nucleic acids may also be employed, or the nucleic acid produced from a previous amplification reaction herein using the same or different primers may be so utilized. The specific nucleic acid sequence to be amplified may be only a fraction of a larger molecule or can be present initially as a discrete molecule, so that the specific sequence constitutes the entire nucleic acid. It is not necessary that the sequence to be amplified be present initially in a pure form; it may be a minor fraction of a complex mixture, such as a portion of the D-globin gene contained in whole human DNA or a portion of nucleic acid sequence due to a particular microorganism which organism might constitute only a very minor fraction of a particular biological sample. The starting nucleic acid may contain more than one desired specific nucleic acid sequence which may be the same or different. Therefore, it is useful not only for producing large amounts of one specific nucleic acid sequence, but also for amplifying simultaneously more than one different specific nucleic acid sequence located on the same or different nucleic acid molecules.

[00124] The nucleic acid or acids may be obtained from any source, for example, from plasmids such as pBR322, from cloned DNA or RNA, or from natural DNA or RNA from any source, including bacteria, yeast, viruses, and higher organisms such as plants or animals. DNA or RNA may be extracted from blood, tissue material such as chorionic villi or amniotic cells.

[00125] It will be understood that the word primer as used hereinafter may refer to more than one primer, particularly in the case where there is some ambiguity in the information regarding the terminal sequence(s) of the fragment to be amplified. For instance, in the case where a nucleic acid sequence is inferred from protein sequence information a collection of primers containing sequences representing all possible codon variations based on degeneracy of the genetic code will be used for each strand. One primer from this collection will be 100% homologous with the end of the desired sequence to be amplified.

[00126] An appropriate agent may be added for inducing or catalyzing the primer extension reaction and the reaction is allowed to occur under conditions known in the art. The inducing agent may be any compound or system which will function to accomplish the synthesis of primer extension products, including enzymes. Suitable enzymes for this purpose may include, for example, E. coli DNA polymerase I, Klenow fragment of E. coli DNA polymerase I, T4 DNA polymerase, other available DNA polymerases, reverse transcriptase, and other enzymes, including heat-stable enzymes, which will facilitate combination of the nucleotides in the proper manner to form the primer extension products which are complementary to each nucleic acid strand. Generally, the synthesis can be initiated at the 3' end of each primer and proceed in the 5' direction along the template strand, until synthesis terminates, producing molecules of different lengths. There may be inducing agents, however, which initiate synthesis at the 5' end and proceed in the other direction, using the same process as described above.

[00127] The newly synthesized strand and its complementary nucleic acid strand form a double- stranded molecule which can be used in the succeeding steps of the process. In the next step, the strands of the double-stranded molecule may be separated to provide single-stranded molecules. New nucleic acid may be synthesized on the single-stranded molecules. Additional inducing agent, nucleotides and primers may be added if necessary for the reaction to proceed under the conditions prescribed above. Again, the synthesis would be initiated at one end of the

oligonucleotide primers and would proceed along the single strands of the template to produce additional nucleic acid. After this step, half of the extension product would consist of the specific nucleic acid sequence bounded by the two primers. The steps of strand separation and extension product synthesis can be repeated as often as needed to produce the desired quantity of the specific nucleic acid sequence. The amount of the specific nucleic acid sequence produced would accumulate in an exponential fashion. After the appropriate length of time has passed to produce the desired amount of the specific nucleic acid sequence, the reaction may be halted by inactivating the enzymes in any known manner or separating the components of the reaction.

[00128] Amplification is useful when the amount of nucleic acid available for analysis is very small, as, for example, in the prenatal diagnosis of sickle cell anemia using DNA obtained from fetal cells. Amplification is particularly useful if such an analysis is to be done on a small sample using non-radioactive detection techniques which may be inherently insensitive, or where radioactive techniques are being employed but where rapid detection is desirable.

[00129] Any known techniques for nucleic acid (e.g., DNA and RNA) amplification can be used with the assays described herein. Preferred amplification techniques are the polymerase chain reaction (PCR) methodologies which comprise solution PCR and in situ PCR.

[00130] The invention is not limited to the use of straightforward PCR. A system of nested primers may be used for example. Other suitable amplification methods known in the field can also be applied such as, but not limited to, ligase chain reaction (LCR), strand displacement amplification (SDA), self- sustained sequence replication (3SR), array based test, and TAQMAN.

[00131] As used herein "amplification" may refer to any in vitro method for increasing the number of copies of a nucleic acid sequence with the use of a DNA polymerase. Nucleic acid amplification results in the incorporation of nucleotides into a DNA molecule or primer thereby forming a new DNA molecule complementary to a DNA template. The newly formed DNA molecule and its template can be used as templates to synthesize additional DNA molecules. As used herein, one amplification reaction may consist of many rounds of DNA replication. DNA amplification reactions include, for example, polymerase chain reactions (PCR). One PCR reaction may consist of 5-100 "cycles" of denaturation, annealing, and synthesis of a DNA molecule.

Fluorescence Microscopy

[00132] Some embodiments of the invention include fluorescence microscopy for a detection of a biological substance in a biological sample. Fluorescence microscopy enables the molecular composition of the structures being observed to be identified through the use of fluorescently- labeled probes of high chemical specificity such as antibodies. It can be done by directly conjugating a fluorophore to a protein and introducing this back into a cell. Fluorescent analogue may behave like the native protein and can therefore serve to reveal the distribution and behavior of this protein in the cell. Along with NMR, infrared spectroscopy, circular dichroism and other techniques, protein intrinsic fluorescence decay and its associated observation of fluorescence anisotropy, collisional quenching and resonance energy transfer are techniques for protein detection. In some cases, microscopy may be used to detect and enumerate cells, such as eosiniophils.

[00133] The naturally fluorescent proteins can be used as fluorescent probes. The jellyfish aequorea victoria produces a naturally fluorescent protein known as green fluorescent protein (GFP). The fusion of these fluorescent probes to a target protein enables visualization by fluorescence microscopy and quantification by fiow cytometry. Without limiting the scope of the present invention, some of the probes are as following:

[00134] Labels: Sensitivity and safety (compared to radioactive methods) of fluorescence has led to an increasing use for specific labeling of nucleic acids, proteins and other biomolecules. Besides fluorescein, other fluorescent labels cover the whole range from 400 to 820 nm. By way of example only, some of the labels are, fluorescein and its derivatives, carboxyfiuoresceins, rhodamines and their derivatives, atto labels, fluorescent red and fluorescent orange: Cy3/Cy5 alternatives, lanthanide complexes with long lifetimes, long wavelength labels - up to 800 nm, DY cyanine labels, and phycobili proteins.

[00135] Conjugates: Antibody conjugates can be generated with specificity for virtually any epitope and are therefore, applicable to imaging a wide range of biomolecules. By way of example only, some of the conjugates are, isothiocyanate conjugates, streptavidin conjugates, and bio tin conjugates.

[00136] Enzyme Substrates: By way of example only, some of the enzyme substrates are fiuoro genie and chromo genie substrates.

[00137] Micro- and Nanoparticles: By way of example only, some of the fluorochromes are: FITC (green fluorescence, excitation/emission = 506/529 nm), rhodamine B (orange

fluorescence, excitation/emission = 560/584 nm), and nile blue A (red fluorescence,

excitation/emission = 636/686 nm). Fluorescent nanoparticles can be used for various types of immunoassays. Fluorescent nanoparticles are based on different materials, such as,

polyacrylonitrile, and polystyrene etc.

[00138] Molecular Rotors: Fluorescent molecular rotors are sensors of micro environmental restriction that become fluorescent when their rotation is constrained. Few examples of molecular constraint include increased dye (aggregation), binding to antibodies, or being trapped in the polymerization of actin. [00139] IEF-Markers: IEF (isoelectric focusing) is an analytical tool for the separation of ampholytes, mainly proteins. An advantage for IEF-Gel electrophoresis with fluorescent IEF- marker is the possibility to directly observe the formation of gradient. Fluorescent IEF-marker can also be detected by UV-absorption at 280 nm (20°C).

[00140] Any or all of these fluorescent probes can be used for the detection of biological substances in the nasal mucus. A peptide library can be synthesized on solid supports and, by using coloring receptors, subsequent dyed solid supports can be selected one by one. If receptors cannot indicate any color, their binding antibodies can be dyed. The method can not only be used on protein receptors, but also on screening binding ligands of synthesized artificial receptors and screening new metal binding ligands as well. Automated methods for HTS and FACS

(fluorescence activated cell sorter) can also be used. A FACS machine originally runs cells through a capillary tube and separate cells by detecting their fluorescent intensities.

Immunoassays

[00141] Some embodiments of the invention include immunoassay for a detection of a biological substance in a biological ample. In immunoblotting like the western blot of electrophoretically separated proteins a single protein can be identified by its antibody. Immunoassay can be competitive binding immunoassay where analyte competes with a labeled antigen for a limited pool of antibody molecules {e.g. radioimmunoassay, EMIT). Immunoassay can be noncompetitive where antibody is present in excess and is labeled. As analyte antigen complex is increased, the amount of labeled antibody-antigen complex may also increase {e.g. ELISA). Antibodies can be polyclonal if produced by antigen injection into an experimental animal, or monoclonal if produced by cell fusion and cell culture techniques. In immunoassay, the antibody may serve as a specific reagent for the analyte antigen.

[00142] Without limiting the scope and content of the present invention, some of the types of immunoassays are, by way of example only, RIAs (radioimmunoassay), enzyme immunoassays like ELISA (enzyme- linked immunosorbent assay), EMIT (enzyme multiplied immunoassay technique), microparticle enzyme immunoassay (MEIA), LIA (luminescent immunoassay), and FIA (fluorescent immunoassay). These techniques can be used to detect biological substances in the nasal specimen. The antibodies - either used as primary or secondary ones - can be labeled with radioisotopes {e.g. 1251), fluorescent dyes {e.g. FITC) or enzymes {e.g. HRP or AP) which may catalyse fluorogenic or luminogenic reactions.

[00143] EMIT (Enzyme Multiplied Immunoassay Technique): EMIT is a competitive binding immunoassay that avoids the usual separation step. A type of immunoassay in which the protein is labeled with an enzyme, and the enzyme-protein-antibody complex is enzymatically inactive, allowing quantitation of unlabelled protein.

[00144] ELISA (Enzyme Linked Immunosorbent Assay): Some embodiments of the invention include ELISA to detect biological substances in the nasal specimen. ELISA is based on selective antibodies attached to solid supports combined with enzyme reactions to produce systems capable of detecting low levels of proteins. It is also known as enzyme immunoassay or EIA. The protein is detected by antibodies that have been made against it, that is, for which it is the antigen. Monoclonal antibodies can be used.

[00145] The test may require the antibodies to be fixed to a solid surface, such as the inner surface of a test tube, and a preparation of the same antibodies coupled to an enzyme. The enzyme may be one {e.g., β-galactosidase) that produces a colored product from a colorless substrate. The test, for example, may be performed by filling the tube with the antigen solution {e.g., protein) to be assayed. Any antigen molecules present may bind to the immobilized antibody molecules. The antibody-enzyme conjugate may be added to the reaction mixture. The antibody part of the conjugate binds to any antigen molecules that were bound previously, creating an antibody-antigen-antibody "sandwich". After washing away any unbound conjugate, the substrate solution may be added. After a set interval, the reaction is stopped {e.g., by adding 1 N NaOH) and the concentration of colored product formed is measured in a spectrophotometer. The intensity of color is proportional to the concentration of bound antigen.

[00146] ELISA can also be adapted to measure the concentration of antibodies, in which case, the wells are coated with the appropriate antigen. The solution {e.g., serum) containing antibody may be added. After it has had time to bind to the immobilized antigen, an enzyme-conjugated anti-immunoglobulin may be added, consisting of an antibody against the antibodies being tested for. After washing away unreacted reagent, the substrate may be added. The intensity of the color produced is proportional to the amount of enzyme-labeled antibodies bound (and thus to the concentration of the antibodies being assayed).

[00147] Radioimmunoassay: Some embodiments of the invention include radioimmunoassays to detect biological substances in the nasal specimen. Radioactive isotopes can be used to study in vivo metabolism, distribution, and binding of small amount of compounds. Radioactive isotopes of 1H, ¹²C, ³¹P, ³²S, and ¹²⁷I in body are used such as ³H, ¹⁴C, ³²P, ³⁵S, and ¹²⁵I.

[00148] In receptor fixation method in 96 well plates, receptors may be fixed in each well by using antibody or chemical methods and radioactive labeled ligands may be added to each well to induce binding. Unbound ligands may be washed out and then the standard can be determined by quantitative analysis of radioactivity of bound ligands or that of washed-out ligands. Then, addition of screening target compounds may induce competitive binding reaction with receptors. If the compounds show higher affinity to receptors than standard radioactive ligands, most of radioactive ligands would not bind to receptors and may be left in solution. Therefore, by analyzing quantity of bound radioactive ligands (or washed-out ligands), testing compounds' affinity to receptors can be indicated.

[00149] The filter membrane method may be needed when receptors cannot be fixed to 96 well plates or when ligand binding needs to be done in solution phase. In other words, after ligand- receptor binding reaction in solution, if the reaction solution is filtered through nitrocellulose filter paper, small molecules including ligands may go through it and only protein receptors may be left on the paper. Only ligands that strongly bound to receptors may stay on the filter paper and the relative affinity of added compounds can be identified by quantitative analysis of the standard radioactive ligands.

[00150] Fluorescence Immunoassays: Some embodiments of the invention include fluorescence immunoassays for a detection of a biological substance in a biological sample. Fluorescence based immunological methods are based upon the competitive binding of labeled ligands versus unlabeled ones on highly specific receptor sites. In some cases, fluorescence immunoassays may be used to detect and enumerate cells, such as eosiniophils.

[00151] The fluorescence technique can be used for immunoassays based on changes in fluorescence lifetime with changing analyte concentration. This technique may work with short lifetime dyes like fluorescein isothiocyanate (FITC) (the donor) whose fluorescence may be quenched by energy transfer to eosin (the acceptor). A number of photoluminescent compounds may be used, such as cyanines, oxazines, thiazines, porphyrins, phthalocyanines, fluorescent infrared-emitting polynuclear aromatic hydrocarbons, phycobiliproteins, squaraines and organo- metallic complexes, hydrocarbons and azo dyes.

[00152] Fluorescence based immunological methods can be, for example, heterogenous or homogenous. Heterogenous immunoassays comprise physical separation of bound from free labeled analyte. The analyte or antibody may be attached to a solid surface. The technique can be competitive (for a higher selectivity) or noncompetitive (for a higher sensitivity). Detection can be direct (only one type of antibody used) or indirect (a second type of antibody is used).

Homogenous immunoassays comprise no physical separation. Double-antibody fluorophore- labeled antigen participates in an equilibrium reaction with antibodies directed against both the antigen and the fluorophore. Labeled and unlabeled antigen may compete for a limited number of anti-antigen antibodies. [00153] Some of the fluorescence immunoassay methods include simple fluorescence labeling method, fluorescence resonance energy transfer (FRET), time resolved fluorescence (TRF), and scanning probe microscopy (SPM). The simple fluorescence labeling method method can be used for receptor-ligand binding, enzymatic activity by using pertinent fluorescence, and as a fluorescent indicator of various in vivo physiological changes such as pH, ion concentration, and electric pressure. TRF is a method that selectively measures fluorescence of the lanthanide series after the emission of other fluorescent molecules is finished. TRF can be used with FRET and the lanthanide series can become donors or acceptors. In scanning probe microscopy, in the capture phase, for example, at least one monoclonal antibody is adhered to a solid phase and a scanning probe microscope is utilized to detect antigen/antibody complexes which may be present on the surface of the solid phase. The use of scanning tunneling microscopy eliminates the need for labels which normally is utilized in many immunoassay systems to detect antigen/antibody complexes.

Nuclear magnetic resonanace (NMR)

[00154] Some embodiments of the invention include NMR for detection of a biological substance in a biological sample. NMR spectroscopy is capable of determining the structures of biological macromolecules like proteins and nucleic acids at atomic resolution. In addition, it is possible to study time dependent phenomena with NMR, such as intramolecular dynamics in macromolecules, reaction kinetics, molecular recognition or protein folding. Heteronuclei like ¹⁵N, ¹³C and ²H, can be incorporated in proteins by uniform or selective isotopic labeling.

Additionally, some new information about structure and dynamics of macromolecules can be determined with these methods.

X-ray crystallography

[00155] Some embodiments of the invention include X-ray crystallography for detection of a biological substance in a biological sample. X-ray crystallography is a technique in which the pattern produced by the diffraction of X-rays through the closely spaced lattice of atoms in a crystal is recorded and then analyzed to reveal the nature of that lattice. This generally leads to an understanding of the material and molecular structure of a substance. The spacings in the crystal lattice can be determined using Bragg's law. X-ray diffraction is commonly carried out using single crystals of a material, but if these are not available, micro crystalline powdered samples may also be used which may require different equipment.

Fluorescence Spectroscopy

[00156] Some embodiments of the invention include fluorescence spectroscopy for detection of a biological substance in a biological sample. By way of example only, conventional fiuorometry is measurement of emission light intensities at defined wavelengths for a certain emission maxima of a fluorophore. Total fluorometry is a collection of data for a continuum of absorption as well as emission wavelengths. Fluorescence polarization is when polarized light is used for excitation and binding of fluorochrome-labeled antigens to specific antibodies. Line narrowing spectroscopy is low-temperature solid-state spectroscopy that derives its selectivity from the narrow-line emission spectra.

[00157] Time-dependent fluorescence spectroscopy comprises time-resolved measurements containing more information than steady-state measurements, since the steady-state values represent the time average of time-resolved determinations. It is a single photon timing technique where the time between an excitation light pulse and the first photon emitted by the sample is measured.

Matrix Assisted Laser Desorption ionization time-of-flight mass spectrometry (MALDI TOF-MS)

[00158] Some embodiments of the invention include MALDI TOF-MS for detection of a biological substance in a biological sample. MALDI TOF-MS provides accurate mass determinations and primary sequence information. Improved mass resolution in MALDI TOF- MS can be obtained by the utilization of a single-stage or a dual-stage reflectron (RETOF-MS). In the reflectron mass spectrum, the isotopic multiplet is well resolved producing a full width half maximum (FWHM) mass resolution of about 3400. Mass resolutions up to 6000 (FWHM) can be obtained for peptides up to about 3000 Da with RETOF-MS. Enhancing the mass resolution can also increase the mass accuracy when determining the ion's mass.

[00159] Both linear and reflectron MALDI-TOF-MS can be utilized for molecular weight determinations of molecular ions and enzymatic digests leading to structural information of proteins. These digests are typically mass analyzed with or without purification prior to molecular weight determinations. Varieties of methodologies have been developed to obtain primary sequence information for proteins and peptides utilizing MALDI TOF-MS. Two different approaches can be taken. The first method is known as protein ladder sequencing and can be employed to produce structurally informative fragments of the analyte prior to insertion into the TOF mass spectrometer and subsequent analysis. The second approach utilizes the phenomenon of metastable ion decay that occurs inside the TOF mass spectrometer to produce sequence information.

[00160] The ladder sequencing with TOF-MS consists of either a time-dependent or

concentration-dependent chemical degradation from either the N- or C-terminus of the protein/peptide into fragments, each of which differs by one amino acid residue. The mixture is mass analyzed in a single MALDI -TOF-MS experiment with mass differences between adjacent mass spectral peaks corresponding to a specific amino acid residue. The order of occurrence in the mass spectrum defines the sequence of amino acids in the original protein/peptide.

[00161] Post-source decay with RETOF-MS MALDI is an ionization technique that produces intact protonated pseudomolecular ion species. A significant degree of metastable ion decay occurs after ion acceleration and prior to detection. The ion fragments produced from the metastable ion decay of peptides and proteins typically include both neutral molecule losses (such as water, ammonia and portions of the amino acid side chains) and random cleavage at peptide bonds. In-source decay with linear TOF-MS is an alternative approach to RETOF-MS for studying metastable ion decay of MALDI generated ions. Primary structural information for peptides and proteins can be obtained by this method. Coherent mass spectral peaks can be produced from these metastable decayed ions giving rise to significant structural information for peptides and proteins.

Surface-enhanced laser desorption ionization - time of flight (SELDI-TOF)

[00162] Some embodiments of the invention include SELDI TOF-MS for detection of a biological substance in a biological sample. This technique utilizes stainless steel or aluminum- based supports, or chips, engineered with chemical (hydrophilic, hydrophobic, pre-activated, normal-phase, immobilized metal affinity, and cationic or anionic) or biological (antibody, antigen binding fragments {e.g. scFv), DNA, enzyme, or receptor) bait surfaces of 1 -2mm in diameter. These varied chemical and biochemical surfaces allow differential capture of proteins based on the intrinsic properties of the proteins themselves. Solubilized tissue or body fluids in volumes as small as 0.1 μΐ can be directly applied to these surfaces, where proteins with affinities to the bait surface may bind. Following a series of washes to remove non-specifically or weakly bound proteins, the bound proteins are laser desorbed and ionized for MS analysis. Masses of proteins ranging from small peptides of less than 1000 Da up to proteins of greater than 300 kDa can be calculated based on time-of- flight. As mixtures of proteins may be analyzed within different samples, a unique sample fingerprint or signature may result for each sample tested. Consequently, patterns of masses rather than actual protein identifications can be produced by SELDI analysis. These mass spectral patterns can be used to differentiate patient samples from one another, such as diseased from normal.

UV-Vis

[00163] Some embodiments of the invention include optical absorption spectroscopy (UV7VIS) for detection of a biological substance in a biological sample. UV7VIS provides light absorption data which helps in the determination of concentration of macromolecules such as, proteins, DNA, nucleotides etc. Organic dyes can be used to enhance the absorption and to shift the absorption into the visible range (e.g. coomassie blue reagents). Resonance raman spectroscopy (RRS) can be used to study molecular structure and dynamics. RRS helps in investigating specific parts of macromolecules by using different excitation wavelengths.

Liquid Chromatography (LC)

[00164] Some embodiments of the invention include LC for a detection of biological substance in a biological sample. Examples of LC are but not limited to, affinity chromatography, gel filtration chromatography, anion exchange chromatography, cation exchange chromatography, diode array-LC and high performance liquid chromatography (HPLC).

[00165] Gel filtration chromatography separates proteins, peptides, and oligonucleotides on the basis of size. Molecules may move through a bed of porous beads, diffusing into the beads to greater or lesser degrees. Smaller molecules may diffuse further into the pores of the beads and therefore move through the bed more slowly, while larger molecules may enter less or not at all and thus move through the bed more quickly. Both molecular weight and three dimensional shapes contribute to the degree of retention. Gel Filtration Chromatography may be used for analysis of molecular size, for separations of components in a mixture, or for salt removal or buffer exchange from a preparation of macromolecules.

[00166] Affinity chromatography is the process of bioselective adsorption and subsequent recovery of a compound from an immobilized ligand. This process allows for the specific and efficient purification of many diverse proteins and other compounds. Ion exchange

chromatography separates molecules based on differences between the overall charges of the proteins. It can be used for the purification of protein, oligonucleotides, peptides, or other charged molecules.

[00167] HPLC can be used in the separation, purification and detection of biological substances in the nasal mucus. Crude tissue extracts may be loaded directly onto the HPLC system and mobilized by gradient elution. Rechromatography under the identical conditions is an option if further purification is warranted or necessary. Reversed phase chromatography (RPC) can be utilized in the process of protein structure determination. HPLC may be coupled with MS. The HPLC method described in Henkin et al, New Frontiers in Immunobiology, 2000, pp. 127-152, is incorporated herein in its entirety.

[00168] The size-exclusion chromatography (SEC) and ion-exchange chromatography (IEC) can be used for separation and purification of biologically active proteins, such as enzymes, hormones, and antibodies. In liquid affinity chromatography (LAC), interaction may be based on binding of the protein due to mimicry of substrate, receptor, etc. The protein may be eluted by introducing a competitive binding agent or altering the protein configuration which may facilitate dissociation. A procedure that can be used in the separation of membrane proteins is the use of nonionic detergents, such as Triton X-100, or protein solubilization by organic solvents with IEC.

[00169] Diode array detector-liquid chromatography (DAD-LC) provides complete, multiple spectra for each HPLC peak, which, by comparison, can provide indication of peak purity. These data can also assign presence of tyr, trp, phe, and possibly others (his, met, cys) and can quantitate these amino acids by 2nd derivative or multi-component analysis. By a post-column derivatization, DAD-LC can also identify and quantitate cys, his and arg in individual peptides. Thus, it is possible to analyze for 6 of the 20 amino acids of each separated peptide in a single LC run, and information can be obtained about presence or absence of these amino acids in a given peptide in a single step. This is assisted by knowing the number of residues in each peptide.

Electrophoresis

[00170] Some embodiments of the invention include electrophoresis for detection of a biological substance in a biological sample. Electrophoresis can be gel electrophoresis or capillary electrophoresis.

[00171] Gel Electrophoresis: Gel electrophoresis is a technique that can be used for the separation of proteins. During electrophoresis, macromolecules are forced to move through pores when an electrical current is applied. Their rate of migration through the electric field depends on strength of the field, size and shape of the molecules, relative hydrophobicity of the samples, and on an ionic strength and temperature of a buffer in which the molecules are moving. After staining, the separated macromolecules in each lane can be seen in a series of bands spread from one end of the gel to the other. Using this technology it is possible to separate and identify protein molecules that differ by as little as a single amino acid. Also, gel electrophoresis allows determination of crucial properties of a protein such as its isoelectric point and approximate molecular weight. Electrofocusing or isoelectric focusing is a technique for separating different molecules by their electric charge differences (if they have any charge). It is a type of zone electrophoresis that takes advantage of the fact that a molecule's charge changes as the pH of its surroundings changes.

[00172] Capillary Electrophoresis: Capillary electrophoresis is a collection of a range of separation techniques which may involve the application of high voltages across buffer filled capillaries to achieve separations. The variations include separation based on size and charge differences between analytes (termed capillary zone electrophoresis (CZE) or free solution CE (FSCE)), separation of neutral compounds using surfactant micelles (micellar electrokinetic capillary chromatography (MECC) or sometimes referred to as MEKC) sieving of solutes through a gel network (capillary gel electrophoresis, GCE), separation of cations (or anions) based on electrophoretic mobility (capillary isotachophoresis, CITP), and separation of zwitterionic solutes within a pH gradient (capillary isoelectric focusing, CIEF). Capillary electrochromatography (CEC) can be an associated electrokinetic separation technique which involves applying voltages across capillaries filled with silica gel stationary phases. Separation selectivity in CEC can be a combination of both electrophoretic and chromatographic processes. Many of the CE separation techniques rely on the presence of an electrically induced flow of solution (electroosmotic flow, EOF) within the capillary to pump solutes towards the detector.

Arrays

[00173] Some embodiments of the invention include arrays for detection of a biological substance in a biological sample. Arrays involve performing parallel analysis of multiple samples against known protein targets. The development of various microarray platforms can enable and accelerate the determination of protein abundance, localization, and interactions in a cell or tissue. Microarrays provide a platform that allows identification of protein interaction or function against a characterized set of proteins, antibodies, or peptides. Protein-based chips array proteins on a small surface and can directly measure the levels of proteins in tissues using fluorescence-based imaging. Proteins can be arrayed on either flat solid phases or in capillary systems (micro fluidic arrays), and several different proteins can be applied to these arrays. In addition to the use of antibodies as array probes, single-stranded oligonucleotides, whose specificity is optimized by in vitro elution (aptamers), offer a viable alternative. Nonspecific protein stains can be then used to detect bound proteins.

[00174] Arrays include, but are not limited to, bead arrays, bead based arrays, bioarrays, bioelectronic arrays, cDNA arrays, cell arrays, DNA arrays, gene arrays, gene expression arrays, frozen cell arrays, genome arrays, high density oligonucleotide arrays, hybridization

arrays, micro cantilever arrays, microelectronic arrays, multiplex DNA hybridization

arrays, nanoarrays, oligonucleotide arrays, oligosaccharide arrays, planar arrays, protein arrays, solution arrays, spotted arrays, tissue arrays, exon arrays, filter arrays, macroarrays, small molecule microarrays, suspension arrays, theme arrays, tiling arrays, and transcript arrays.

Sensors

[00175] Some embodiments of the invention include sensors for detection of a biological substance in a biological sample. Sensors can be used for both in vivo and in vitro detection. Sensors can be chemical sensors, optical sensors, and biosensors. Chemical sensors are miniaturized analytical devices which may deliver real-time and online information on the presence of specific compounds or ions in complex samples. Optical sensors are based on measurement of either intrinsic optical properties of analytes, or of optical properties of indicator dyes or labeled biomolecules attached to solid supports. Biosensors can be affinity biosensor based on capabilities of enzymes to convert substrates into products or catalytic biosensors. Biosensors detect antibody and analyte complexes using a variety of physical methods. Some biosensors measure the change in surface charge that occurs when analyte is bound to antibodies or other binding agents, which in turn are bound to a surface. Other biosensors use binding agents attached to a surface and measure a change in a physical property of the support, other than surface charge, upon binding of analyte. Some biosensor techniques use a specific property of a labeled binding agent or antigen to produce a measurable change.

Methods for Identifying Proteins from a Library Screen

[00176] Protein identification methods by way of example only include low-throughput sequencing through Edman degradation, mass spectrometry techniques, peptide mass

fingerprinting, de novo sequencing, and antibody-based assays. The protein quantification assays include fluorescent dye gel staining, tagging or chemical modification methods (i.e.

isotope-coded affinity tags (ICATS), combined fractional diagonal chromatography

(COFRADIC)). The purified protein may also be used for determination of three-dimensional crystal structure, which can be used for modeling intermolecular interactions. Common methods for determining three-dimensional crystal structure include x-ray crystallography and NMR spectroscopy. Detailed below are a few of the methods for identifying proteins in the present invention.

[00177] Protein sequencing: N-terminal sequencing aids in the identification of unknown proteins, confirm recombinant protein identity and fidelity (reading frame, translation start point, etc.), aid the interpretation of NMR and crystallo graphic data, demonstrate degrees of identity between proteins, or provide data for the design of synthetic peptides for antibody generation, etc. N-terminal sequencing utilises the Edman degradative chemistry, sequentially removing amino acid residues from the N-terminus of the protein and identifying them by reverse-phase HPLC. Sensitivity can be at the level of 100s femtomoles and long sequence reads (20-40 residues) can often be obtained from a few 10s picomoles of starting material. Pure proteins (>90%) can generate easily interpreted data, but insufficiently purified protein mixtures may also provide useful data, subject to rigorous data interpretation. N-terminally modified (especially acetylated) proteins cannot be sequenced directly, as the absence of a free primary amino-group prevents the Edman chemistry. However, limited proteolysis of the blocked protein (e.g. using cyanogen bromide) may allow a mixture of amino acids to be generated in each cycle of the instrument, which can be subjected to database analysis in order to interpret meaningful sequence information. C-terminal sequencing is a post-translational modification, affecting the structure and activity of a protein. Various disease situations can be associated with impaired protein processing and C-terminal sequencing provides an additional tool for the investigation of protein structure and processing mechanisms.

[00178] Proteome analyses: Proteomics can be identified primarily by computer search algorithms that assign sequences to a set of empirically acquired mass/intensity data which are generated from conducting electrospray ionization (ESI), matrix-assisted laser

desorption/ionization (MALDI-TOF), or three-dimensional quadrupole ion traps on the protein of interest.

DIAGNOSIS

[00179] Generally, the composition and methods of this disclosure provide for the diagnosis or treatment of smell loss (e.g. , hyposmia, anosmia) and/or taste loss (e.g. , hypogeusia, ageusia) by detecting one or more biological substances.

Examples of Biological Substances

[00180] Various substances that can be analyzed and/or measured in the methods disclosed herein include, by way of example only, proteins, carbohydrates, lipids, hormones (e.g., leptin, ghrelin) in control of appetite, cholesterol and other lipids and lipid carrying proteins in control of lipid metabolism, growth factors (e.g. , hepatic growth factor, granulocyte colony growth factor, brain derived neurotrophic factor), and antibodies, liver enzymes (SGOT, SGPT) therapeutic and recreational drugs of abuse, trace metals [either excess as in toxicity (e.g. , lead, mercury, arsenic) or in deficiency diseases involving zinc, copper, magnesium] and most other substances found in plasma, erythrocytes, urine and saliva. Each metabolite in nasal mucus may reflect both physiological and pathological changes in human body metabolism specific to each metabolite and may reflect the manner in which nasal mucus provides information both on human body metabolism such as provided by plasma, erythrocytes, urine and saliva or information relatively unique to nasal mucus.

[00181] Biological substances can comprise cytokines, such a pro-inflammatory cytokines or anti- inflammatory cytokines. Pro -inflammatory cytokines can include IL-la, IL-Ι β, IL-6, IL-18, TNF-a, or a combination thereof. Anti- inflammatory cytokins can include IL-lra, IL-10, ΙΚΝ-γ, or a combination thereof. The balance of pro- and anti-inflammatory cytokines can indicate whether a subject has a smell or taste disorder. [00182] Biological substances can comprise cytokine receptors such as type I cytokine receptors, type II cytokine receptors, members of the immunoglobulin superfamily, members of the tumor necrosis factor receptor family, chemokine receptros, and or TGF beta receptors. In some embodiments, a cytokine receptor is IL-1 RII and/or IL-2R.

[00183] Biological substances can comprise eosinophils. Biological substances can comprise IgE protein. Biological substances can comprise cyclic nucleotides (e.g., cAMP, cGMP). Biological substances can comprise nitric oxide (NO).

[00184] The identification and analysis of biological substances as disclosed herein has numerous therapeutic and diagnostic applications. Clinical applications include, for example, detection of taste or smell disorder; distinguishing the underlying cause of the taste or smell disorders to inform prognosis, selection of therapy, and/or prediction of therapeutic response; monitoring of therapy associated with efficacy and toxicity; and detection of recurrence of the taste or smell disorders.

[00185] The presence or increase or decrease of biological substances' concentration can allow the physician diagnose taste or smell disorders and/or to predict the efficacy of treatment regimes.

[00186] The diagnosis of taste or smell disorders as disclosed herein can be used to enable or assist in the pharmaceutical drug development process for therapeutic agents. The analysis can be used to diagnose patients enrolling in a clinical trail. The diagnosis can indicate the state of the taste or smell disorders in patients undergoing treatment in clinical trials, and show changes in the state during the treatment. The diagnosis can demonstrate the efficacy of a treatment, and can be used to stratify patients according to their responses to various therapies.

[00187] The methods of the present invention can be used to evaluate the efficacy of treatments over time. For example, biological samples can be obtained from a patient over a period of time as the patient is undergoing treatment. The biological substances from the different samples can be compared to each other to determine the efficacy of the treatment. Also, the methods described herein can be used to compare the efficacies of different therapies and/or responses to one or more treatments in different populations (e.g. , different age groups, ethnicities, family histories, cause of taste or smell loss, etc.).

General Methods for Diagnosis

[00188] Generally, the compositions and methods of this disclosure provide for evaluating a subject's olfactory function by determining a detection threshold (DT) score, a recognition threshold (RT) score, a magnitude estimation (ME) score, a hedonic (H) score, or a combination thereof. These score may be determined as previously described in Henkin RI, Levy LM, and Fordyce A. Taste and smell function in chronic disease: A review of clinical and biochemical evaluations of taste and smell dysfunction in over 5000 patients at The Taste and Smell Clinic in Washington, DC. Am J Otolaryngol. 2013 Sep-Oct;34(5):477-89 (and incorporated by reference herein).

[00189] For example patients may be initially diagnoses with suspected hyposomia, if their sensory dysfunction manifests as either loss of taste (i.e., flavor) and/or smell function. This subjective response may be documented by objective psychophysical measurements of olfactory function administered to each patient by use of a forced-choice, three-stimuli, stepwise-staircase technique in a fixed, controlled design as previously described herein and in Henkin, R.I.

Evaluation and treatment of human olfactory dysfunction, in Otolaryngology (English, G.M. Ed.), Lippincott, Philadelphia, 1993, Vol.2, pp.1-86 ( incorporated by reference herein)

[00190] In some cases, four test odors are used; they may be pyridine (dead-fish odor), nitrobenzene (bitter-almond odor), thiophene (petroleum-like odor) and amyl acetate (banana-oil odor). Detection thresholds (DT), recognition thresholds (RT) and magnitude estimation (ME) values for each odor may be determined as previously described. Thresholds may be converted into bottle units (BU) as previously described (53) and results reported as M±SEM of correct responses for each odor in each treatment group; ME may be reported in % and results calculated to obtain M±SEM for each treatment group for all correct responses using data for the four highest odor concentrations presented (from 10^"2M - an absolute odor concentration).

[00191] In addition, each patient may be graded using the hedonic (H) value of each odor presented for these same odor concentrations (from 10^"2M - an absolute odor concentration using a -100 - 0 - +100 scale). If they consider a pleasant odor pleasant ("they wished to smell the odor again") they may be graded the odor as +1 - +100 with respect to pleasantness; if they consider the odor unpleasant ("they did not wish to smell the odor again") they graded the odor as -1 - -100 with respect to unpleasantness; if they do not consider the odor either pleasant or unpleasant they may be graded the odor as neutral or 0. Results may be obtained by calculating the arithmetical sum of each correct recognition response for each odor with respect to its pleasantness, unpleasantness or neutrality. Arithmetic M+SEM may be obtained for each treatment group for each odor presented. These score may then be compared to a reference or threshold levels. This comparison may be used in aiding the diagnosis of hyposmia.

Biomarkers for diagnosing taste and smell disorders

[00192] Disclosed herein are biomarkers and methods for diagnosing a taste or smell disorder in a subject. [00193] In some methods of diagnosing a taste or smell disorder in a subject, the methods comprise: (a) measuring a level of one or more pro-inflammatory cytokines selected from the group consisting of IL-la, IL-Ι β, IL-6, IL-18 in a biological sample from the subject; and (b) diagnosing the subject with the taste or smell disorder based upon the level of one or more proinflammatory cytokines that is higher than a threshold level.

[00194] In some embodiments, the threshold level is an average level of the one or more proinflammatory cytokines as measured in a control population. In some embodiments, the threshold level is at least 1.5 times an average level of the one or more pro-inflammatory cytokines as measured in a control population.

[00195] In some embodiments, the biological sample comprises a blood sample, a plasma sample, a urine sample, a saliva sample, a nasal mucus sample, or a combination thereof. In some embodiments, the biological sample comprises a plasma sample, a urine sample, a saliva sample, or a nasal mucus sample.

[00196] In some embodiments, measuring comprises using one or more antibodies that bind the one or more pro-inflammatory cytokines. In some embodiments, at least one antibody is conjugated to an enzyme, a fluorescent molecule, or a radio-label. In some embodiments, the one or more antibodies are used in an immunostain, an immunoprecipitation, an

[00197] Some embodiments further comprise measuring a level of one or more antiinflammatory cytokines in the biological samples. In some embodiments, the one or more antiinflammatory cytokines comprise IL-lra, IL-10, IFN-γ, or a combination thereof. In some embodiments, diagnosing is further based upon the level of at least one anti-inflammatory cytokine being lower than a second threshold level. In some embodiments, the second threshold level is an average anti-inflammatory cytokine level as measured in a control population. In some embodiments, the second threshold level is at least 1.5 times lower than an average antiinflammatory cytokine level as measured in a control population.

[00198] In some methods of diagnosing a taste or smell disorder in a subject, the methods comprise: (a) measuring a level of IL-6 in a biological sample from the subject; (b) diagnosing the subject with the taste or smell disorder based upon the level of IL-6 that is higher than a threshold level. [00199] In some embodiments, the threshold level is an average level of the IL-6 as measured in a control population. In some embodiments, the threshold level is at least 1.5 times an average level of the IL-6 as measured in a control population.

[00200] In some embodiments, the biological sample comprises a blood sample, a plasma sample, a urine sample, a saliva sample, a nasal mucus sample, or a combination thereof. In some embodiments, the biological sample comprises a plasma sample, a urine sample, a saliva sample, or a nasal mucus sample.

[00201] In some embodiments, the biological sample is a nasal mucus sample. In some embodiments, the threshold level is from 5 pg/mL to about 15 pg/mL.

[00202] In some embodiments, the biological sample is a plasma sample. In some embodiments, the threshold level is from 0.05 pg/mL to about 0.2 pg/mL.

[00203] In some embodiments, the biological sample is a saliva sample. In some embodiments, the threshold level is from 0.15 pg/mL to about 0.4 pg/mL.

[00204] In some embodiments, measuring comprises using an antibody that binds IL-6. In some embodiments, the antibody is conjugated to an enzyme, a fluorescent molecule, or a radio-label. In some embodiments, the antibody is used in an immunostain, an immunoprecipitation, an Immunoelectrophoresis, an immunoblot, a western blot, a proximity ligation assay, or a spectrophotometry assay. In some embodiments, the antibody is used in the spectrophotometry assay that is an EMIT (Enzyme Multiplied Immunoassay Technique) assay, an ELISA (Enzyme Linked Immunosorbent Assay), a sandwich ELISA, or a competitive ELISA.

[00205] In some methods of diagnosing a taste or smell disorder in a subject, the methods comprise: (a) measuring a level of IgE protein in a biological sample from the subject; and (b) diagnosing the subject with the taste or smell disorder based upon the level of IgE protein that is higher than a threshold level.

[00206] In some embodiments, the threshold level is an average level of IgE protein as measured in a control population. In some embodiments, the threshold level is at least 1.5 times an average level of IgE protein as measured in a control population.

[00207] In some embodiments, the biological sample comprises a blood sample, a plasma sample, a urine sample, a saliva sample, a nasal mucus sample, or a combination thereof. In some embodiments, the biological sample comprises a blood sample or a plasma sample. In some embodiments, the threshold level is from 75 kU/L to 125 kU/L. In some embodiments, the threshold level is about: 75 kU/L, 100 kU/L, or 125 kU/L. In some embodiments, measuring the level of IgE protein comprises a fluorescence polarization assay. [00208] In some methods of diagnosing a taste or smell disorder in a subject, the methods comprise: (a) measuring a level of eosiniphils in a biological sample from the subject; and (b) diagnosing the subject with the taste or smell disorder based upon the level of eosinophils that is higher than a threshold level.

[00209] In some embodiments, the threshold level is an average level of eosinophils as measured in a control population. In some embodiments, the threshold level is at least 1.5 times an average level of eosinophils as measured in a control population.

[00210] In some embodiments, the biological sample comprises a blood sample, a plasma sample, a urine sample, a saliva sample, a nasal mucus sample, or a combination thereof. In some embodiments, the biological sample comprises a blood sample or a plasma sample. In some embodiments, the threshold level is from 300 cells/HPF (high powered field) to 400 cells/HPF. In some embodiments, the threshold level is about: 300 cells/HPF, 350 cells/HPF, or 400 cells/HPF. In some embodiments, measuring the level of eosiniphils is performed with a Coulter counter.

[00211] In some methods of diagnosing a taste or smell disorder in a subject, the methods comprise: (a) measuring a level of one or more proteins selected from the group consisting of IL- la, IL-Ιβ, IL-lra, IL-1 RII, IL-2, IL-2R, IL-6, IL-10, IL-18, IFN-β, and IFN-γ in a nasal mucus sample from the subject; and (b) diagnosing the subject with the taste or smell disorder based upon one or more measurements comprising: (i) the level of IL-1 a that is about: 125 pg/mL to 195 pg/mL, 150 pg/mL to 170 pg/mL, 120 pg/mL to 170 pg/mL, or 150 pg/mL to 195 pg/mL; (ii) the level of IL-Ιβ that is about: 195 pg/mL to 300 pg/mL, 220 pg/mL to 275 pg/mL, 220 pg/mL to 300 pg/mL, or 195 pg/mL to 275 pg/mL; (iii) the level of IL-lra that is about: 30,000 pg/mL to 90,000 pg/mL, 45,000 pg/mL to 75,000 pg/mL, 45,000 pg/mL to 90,000 pg/mL, or 30,000 pg/mL to 75,000 pg/mL; (iv) the level of IL-1 RII that is about: 960 pg/mL to 2600 pg/mL, 1370 pg/mL to 2190 pg/mL, 1370 pg/mL to 2600 pg/mL, or 960 pg/mL to 2190 pg/mL; (v) the level of IL-2 that is about: 0 pg/mL, 0.1 pg/mL, 0.2 pg/mL, 0.3 pg/mL, or 0.5 pg/mL; (vi) the level of IL-2R that is about: 0 to 200 pg/mL, 50 to 150 pg/mL, 0 to 150 pg/mL, or 50 to 200 pg/mL; (vii) the level of IL-6 that is about: 0.1 pg/mL to 2.2 pg/mL, 0.6 pg/mL to 1.7 pg/mL, 0.6 pg/mL to 2.2 pg/mL, or 0.1 pg/mL to 1.7 pg/mL; (viii) the level of IL-10 that is about: 0 pg/mL to 3.5 pg/mL, 0.8 pg/mL to 2.7 pg/mL, 0.8 pg/mL to 3.5 pg/mL, or 0 pg/mL to 2.7 pg/mL; (ix) the level of IL-18 that is about: 40 pg/mL to 290 pg/mL, 100 pg/mL to 230 pg/mL, 40 pg/mL to 230 pg/mL, or 100 pg/mL to 290 pg/mL; (x) the level of IFN-β that is about: 0 pg/mL to 910 pg/mL, 230 pg/mL to 680 pg/mL, 230 pg/mL to 910 pg/mL, or 0 pg/mL to 680 pg/mL; or (xi) the level of IFN-γ that is about: 55 pg/mL to 110 pg/mL, 70 pg/mL to 95 pg/mL, 70 pg/mL to 110 pg/mL, 55 pg/mL to 95 pg/mL. In some embodiments, diagnosing is based upon 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 of the one or more measurements.

[00212] In some embodiments, measuring comprises using one or more antibodies that bind the one or more proteins. In some embodiments, at least one antibody is conjugated to an enzyme, a fluorescent molecule, or a radio-label. In some embodiments, the one or more antibodies are used in an immunostain, an immunoprecipitation, an Immunoelectrophoresis, an immunoblot, a western blot, a proximity ligation assay, or a spectrophotometry assay. In some embodiments, the one or more antibodies are used in the spectrophotometry assay that is an EMIT (Enzyme Multiplied Immunoassay Technique) assay, an ELISA (Enzyme Linked Immunosorbent Assay), a sandwich ELISA, or a competitive ELISA.

[00213] In any of the diagnostic methods disclosed herein, the taste or smell disorder can be anosmia, hyposmia, phantosmia, ageusia, hypogeusia, or phantageusia. In some embodiments, the taste or smell disorder is anosmia or hyposmia.

[00214] Any of the diagnostic methods disclosed herein can further comprise measuring a level of one or more of cAMP, cGMP, or nitric oxide (NO) in a sample from the subject. In some embodiments, diagnosing is further based on the level of cAMP, cGMP, and/or nitric oxide (NO) that is lower than an average level measured in a control population.

[00215] In any of the diagnostic methods disclosed herein, measuring can comprise using one or more techniques that are fluorescence microscopy, a radioimmunoassay, a fluorescence immunoassay, fluorescence-activated cell sorting, mass spectrometry, liquid chromatography, electrophoresis, protein arrays, or a combination thereof.

[00216] In any of the diagnostic methods disclosed herein, the subject diagnosed with the taste or smell disorder can be further identified as a member of an orphan drug population.

[00217] Any of the diagnostic methods disclosed herein can further comprise evaluating the subject's taste or smell function by determining a detection threshold (DT) score, a recognition threshold (RT) score, a magnitude estimation (ME) score, a hedonic (H) score, or a combination thereof with a forced-choice, three-stimuli, stepwise-staircase technique using one or more tastants or odorants. In some embodiments, smell function is tested using the one or more odorants that comprise pyridine, nitrobenzene, thiophene, amyl acetate, or a combination thereof. In some embodiments, taste function is tested using the one or more tastants that comprise sodium chloride (NaCl), sucrose, hydrogen chloride (HC1), urea, or a combination thereof. In some embodiments, diagnosing is further based upon the DT score that is higher than an average DT score as measured in a control population. In some embodiments, diagnosing is further based upon the RT score that is higher than an average RT score as measured in a control population. In some embodiments, diagnosing is further based upon the ME score that is lower than an average ME score as measured in a control population. In some embodiments, diagnosing is further based upon the H score that is different than an average H score as measured in a control population.

[00218] Additionally, the present disclosure also provides for the measurement of levels of one or more biological substances associated with hyposmia. These levels may also be compared to a threshold level, wherein the comparison is also used to aid in the diagnosis of hyposmia. In some cases levels of these biological substances may include but are not limited to IL-l , IL-Ιβ, IL-lra, IL-1 RII, IL-la, IL-Ιβ, IL-lra, IL-1 RII, IL-2, IL-2R, IL-6, IL-10, IL-18, TNF-a, IFN-β, IFN-γ, cytokines, IgE, or eosinophils.

[00219] For example, in some cases, molecules such as is IL-la, IL-Ιβ, IL-lra, IL-1 RII, IL-la, IL-Ιβ, IL-lra, IL-1 RII, IL-2, IL-2R, IL-6, IL-10, IL-18, TNF-a, IFN-β, IFN-γ, or a combination thereof may be elevated. In some cases the molecule is IL-6. In some cases the threshold for determining elevation about 15 pg/mL to about 45 pg/mL and wherein higher levels of the molecules described herein indicate that the subject has hyposmia. In some cases, the thresh hold may be 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 50, 60, 70 80, 90 or 100 pg/mL.

[00220] In some cases, measuring or testing biological substances, such as cytokine, IgE protein, and/or eosinophils, IL-la, IL-Ιβ, IL-lra, IL-1 RII, IL-la, IL-Ιβ, IL-lra, IL-1 RII, IL-2, IL-2R, IL-6, IL-10, IL-18, TNF-a, IFN-β, IFN-γ, cAMP, cGMP, nitric oxide or combination thereof, may be used to identify orphan drug population patients. In some cases, a candidate drug may be tested in a population of subjects, or a subpopulation of subjects, such as an orphan drug subpopulation. In some cases, the measurement or testing of the biological substances, as described herein, may be used to determine the efficacy of an orphan drug candidate. For example, in the case IL-6, an orphan drug candidate may be identified if IL-6 levels decrease in a subject after administration of the drug candidate. In some cases, the change in level of the biological substance may correlate with amelioration of other symptoms of the hyposmia, anosmia, hypogeusia or ageusia or other disease.

[00221] In some cases, measurements or testing of one or more biological substances may be compared to thresholds or may be compared to level or amounts of other biological substances.

[00222] For example, in some cases, elevated levels of IgE in blood samples may be used in aiding the diagnosis of hyposmia. In some cases the threshold level may be 75 kU/L. In some cases, the threshold may be 75 kU/L-125 kU/L. In some cases, the threshold hold may be 45, 55, 65, 75, 76, 77, 78, 79, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 150, 175, 200 kU/L. In some cases, elevated level of IgE may be elevated above a threshold value. Elevated levels may indicate that a subject has a taste or smell disorder. In some cases, a subject may have a taste or smell disorder if the subject's IgE values are measured above 45, 55, 65, 75, 76, 77, 78, 79, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 150, 175, 200 kU/L. A subject's IgE value may be determined by any suitable assay as described herein. In some embodiments, an IgE level can be meausured in a fluorescence polarization assay.

[00223] For example, in some cases, elevated levels of eosiniophils in blood samples may be used in aiding the diagnosis of taste or smell disorders. In some cases the threshold level may be 200 cells/HPF. In some cases, the threshold may be 200 cells/HPF-400 cells/HPF. In some cases the threshold may be 50-600 cells/HPF. In some cases, the threshold hold may be 50, 100, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390 or 400, 420, 440, 460, 480, 500, 520, 540, 560, 580 or 600 cells/HPF. In some cases, elevated level of eosiniophils may be elevated about a threshold value. Elevated levels may indicate that a subject has a taste or smell disorder. In some cases, a subject may have the taste or smell disorder if the subject's eosiniophil count or levels may be measured above 50, 100, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390 or 400, 420, 440, 460, 480, 500, 520, 540, 560, 580 or 600 cells/HPF. A subject's eosiniophil count may be determined by any suitable assay as described herein, including microscopy or Coulter counter.

[00224] In one aspect, disclosed herein are methods of diagnosing hyposmia, anosmia, hypogeusia, or ageusia in a subject, the methods comprising: (a) obtaining one or more biological samples from the subject; (b) measuring a level of one or more pro-inflammatory cytokines in at least one of the one or more biological samples; and (c) diagnosing the subject with hyposmia, anosmia, hypogeusia, or ageusia based upon the level of one or more proinflammatory cytokines that is higher than a threshold level. In some embodiments, the threshold level is an average level for the one or more pro-inflammatory cytokines as measured in a control population comprising subjects with normal olfactory and/or taste function.

[00225] In some embodiments, the one or more pro -inflammatory cytokines comprise IL-l , IL- 1β, IL-6, IL-18, TNF-a, or a combination thereof. In some embodiments, the one or more proinflammatory cytokines comprise IL-6. In some embodiments, the threshold level for IL-6 is about 4 pg/mL to about 12 pg/mL.

[00226] Some embodiments further comprise measuring a level of one or more antiinflammatory cytokines in at least one of the biological samples. In some embodiments, the one or more anti- inflammatory cytokines comprise IL-lra, IL-10, IFN-γ, IFN-β, or a combination thereof. In some embodiments, diagnosing is further based upon the level of at least one of the one or more anti- inflammatory cytokines being lower than an average anti- inflammatory cytokine level as measured in the control population comprising subjects with normal olfactory function.

[00227] Some embodiments further comprise measuring a level of IgE protein, eosinophils, cAMP, cGMP, nitric oxide (NO), IL-1 RII, IL-2R, or a combination thereof in at least one of the one or more biological samples.

[00228] In some embodiments, diagnosing is further based upon the level of IgE protein being higher than an average IgE level as measured in the control population comprising subjects with normal olfactory function. In some embodiments, diagnosing is further based upon the level of IgE protein being higher than about: 75 kU/L, 100 kU/L, or 125 kU/L. In some embodiments, measuring the level of IgE protein comprises a fluorescence polarization assay.

[00229] Some embodiments further comprise measuring the level of eosinophils, wherein diagnosing is further based upon the level of eosinophils being higher than an average eosinophils level as measured in the control population comprising subjects with normal olfactory function. In some embodiments, diagnosing is further based upon the level of eosinophils being higher than about 300 cells/HPF (high powered field), 350 cells/HPF, or 400 cells/HPF. In some embodiments, measuring the level of eosiniphils is performed with a Coulter counter.

[00230] Some embodiments further comprise measuring the level of NO, wherein diagnosing is further based upon the level of NO being lower than a threshold value (e.g., an average NO level as measured in a control population, e.g., comprising subjects with normal olfactory function).

[00231] Some embodiments further comprise measuring the level of cAMP, wherein diagnosing is further based upon the level of cAMP being lower than a threshold value (e.g., an average cAMP level as measured in a control population, e.g., comprising subjects with normal olfactory function).

[00232] Some embodiments further comprise measuring the level of cGMP, wherein diagnosing is further based upon the level of cGMP being lower than a threshold value (e.g., an average cGMP level as measured in the control population, e.g., comprising subjects with normal olfactory function).

[00233] Some embodiments further comprise evaluating the subjects olfactory function by determining a detection threshold (DT) score, a recognition threshold (RT) score, a magnitude estimation (ME) score, or a combination thereof with a forced-choice, three-stimuli, stepwise- staircase technique using one or more olfaction testing compounds. In some embodiments, the one or more olfaction testing compounds comprise pyridine, nitrobenzene, thiophene, amyl acetate, or a combination thereof. In some embodiments, diagnosing is further based upon the DT score being higher than an average DT score as measured in the control population comprising subject with normal olfactory function, the RT score being higher than an average RT score as measured in the control population comprising subject with normal olfactory function, and/or the ME score being lower than an average ME score as measured in the control population comprising subject with normal olfactory function.

[00234] In some embodiments, the one or more biological samples comprise a blood sample, a plasma sample, a urine sample, a saliva sample, a nasal mucus sample, or a combination thereof. In some embodiments, the one or more biological samples comprise the nasal mucus sample.

[00235] In some embodiments, measuring comprises using one or more antibodies that bind the one or more pro-inflammatory cytokines. In some embodiments, the one or more antibodies are used in an immunostain, an immunoprecipitation, an Immunoelectrophoresis, an immunoblot, a western blot, or a spectrophotometry assay. In some embodiments, the one or more antibodies are used in the spectrophotometry assay that is an EMIT (Enzyme Multiplied Immunoassay Technique) assay or an ELISA (Enzyme Linked Immunosorbent Assay). In some embodiments, measuring comprises using one or more techniques that are fluorescence microscopy, a radioimmunoassay, a fluorescence immunoassay, mass spectrometry, liquid chromatography, electrophoresis, or a combination thereof.

[00236] Some embodiments further comprise treating the taste or smell disorder in the subject diagnosed with hyposmia, anosmia, hypogeusia, or ageusia that is a subject in need thereof. In some embodiments, treating comprises administering a steroid-free pharmaceutical dosage unit for intranasal administration comprising a positive amount of one or more PDE inhibitors effective for treating hyposmia, anosmia, hypogeusia, or ageusia to the subject in need thereof. In some embodiments, the steroid-fee pharmaceutical dosage unit comprises the positive amount that is less than 45 mg, 30 mg, 15 mg, 10 mg, 5 mg, 1 mg, 500 μg, 250 μg, 120 μg, 80 μg, 40 μg, or 20 μg individually of the one or more PDE inhibitors and a pharmaceutically acceptable carrier.

[00237] In some embodiments, the one or more PDE inhibitors comprise a non-selective PDE inhibitor, a PDE-1 selective inhibitor, a PDE-2 selective inhibitor, a PDE-3 selective inhibitor, a PDE-4 selective inhibitor, a PDE-5 selective inhibitor, a PDE- 10 selective inhibitor, or a combination thereof. Some embodiments comprise the non-selective PDE inhibitor that is a methylxanthine derivative. In some embodiments, the methylxanthine derivative is caffeine, theophylline, IBMX (3-isobutyl-l-methylxanthine) aminophylline, doxophylline, cipamphylline, neuphylline, pentoxiphylline, or diprophylline. In some embodiments, the methylxanthine derivative is theophylline. Some embodiments comprise the PDE 1 inhibitor that is vinpocetine. Some embodiments comprise the PDE 2 inhibitor that is EHNA. Some embodiments comprise the PDE 3 inhibitor that is inamrinone, anagrelide, or cilostazol. Some embodiments comprise the PDE 4 inhibitor that is mesembrine, rolipram, ibudilast, piclamilast, luteolin, drotaverine, or roflumilast. Some embodiments comprise the PDE 5 inhibitor that is sildenafil, tadalafil, vardenafil, udenafil, avanafil, or dipyridamole. Some embodiments comprise the PDE 10 inhibitor that is papaverine.

[00238] In some embodiments, treating comprises intranasal administration of an effective amount of one or more anti-inflammatory cytokines. In some embodiments, the one or more anti- inflammatory cytokines comprise IL-lra, IL-10, IFN-γ, IFN-β, or a combination thereof.

[00239] In some embodiments, treating comprises intranasal administration of an effective amount of an antibody, antibody fragment, or antibody mimetic that inhibits one of the one or more pro -inflammatory cytokines. In some embodiments, the pro-inflammatory cytokine is IL- l , IL-Ιβ, IL-6, IL-18, TNF-a, or a combination thereof. In some embodiments, the antibody, antibody fragment, or antibody mimetic binds to one of the one or more pro -inflammatory cytokines. In some embodiments, the antibody, antibody fragment, or antibody mimetic binds to a receptor for at least one of the one or more pro -inflammatory cytokines. Some embodiments comprise intranasal administration of the antibody that is a monoclonal antibody. In some embodiments, the monoclonal antibody is a recombinant antibody, a chimeric antibody, a human monoclonal antibody, or a humanized monoclonal antibody. Some embodiments comprise administration of the antibody fragment that is a FAB fragment, a FAB2 fragment, a Fv fragment, a ScFv fragment, an antibody light chain, or an antibody heavy chain. Some embodiments comprise administration of the antibody mimetic that is an affibody molecule, an affilin, an affitin, an anticalins, an avimers, a DARPins, a fynomer, a Kunitz domain peptide, or a monobody.

[00240] In another aspect, a profile of cytokines and cytokine receptors is provided that enable the identification and diagnosis of a subject with hyposmia, anosmia, hypogeusia, and/or ageusia. Accordingly, disclosed herein are methods of diagnosing hyposmia or anosmia in a subject, the methods comprising: (a) obtaining a nasal mucus sample from the subject; (b) measuring a level of one or more of IL-la, IL-Ιβ, IL-lra, IL-1 RII, IL-2, IL-2R, IL-6, IL-10, IL- 18, IFN-β, or IFN-γ in the nasal mucus sample; and (c) diagnosing the subject with hyposmia or anosmia based upon one or more measurements comprising: (i) the level of IL-la that is about: 125 pg/mL to 195 pg/mL, 150 pg/mL to 170 pg/mL, 120 pg/mL to 170 pg/mL, or 150 pg/mL to 195 pg/mL; (ii) the level of IL-Ιβ that is about: 195 pg/mL to 300 pg/mL, 220 pg/mL to 275 pg/mL, 220 pg/mL to 300 pg/mL, or 195 pg/mL to 275 pg/mL; (iii) the level of IL-lra that is about: 30,000 pg/mL to 90,000 pg/mL, 45,000 pg/mL to 75,000 pg/mL, 45,000 pg/mL to 90,000 pg/mL, or 30,000 pg/mL to 75,000 pg/mL; (iv) the level of IL-1 RII that is about: 960 pg/mL to 2600 pg/mL, 1370 pg/mL to 2190 pg/mL, 1370 pg/mL to 2600 pg/mL, or 960 pg/mL to 2190 pg/mL; (v) the level of IL-2 that is about: 0 pg/mL, 0.1 pg/mL, 0.2 pg/mL, 0.3 pg/mL, or 0.5 pg/mL; (vi) the level of IL-2R that is about: 0 to 200 pg/mL, 50 to 150 pg/mL, 0 to 150 pg/mL, or 50 to 200 pg/mL; (vii) the level of IL-6 that is about: 0.1 pg/mL to 2.2 pg/mL, 0.6 pg/mL to 1.7 pg/mL, 0.6 pg/mL to 2.2 pg/mL, or 0.1 pg/mL to 1.7 pg/mL; (viii) the level of IL-10 that is about: 0 pg/mL to 3.5 pg/mL, 0.8 pg/mL to 2.7 pg/mL, 0.8 pg/mL to 3.5 pg/mL, or 0 pg/mL to 2.7 pg/mL; (ix) the level of IL-18 that is about: 40 pg/mL to 290 pg/mL, 100 pg/mL to 230 pg/mL, 40 pg/mL to 230 pg/mL, or 100 pg/mL to 290 pg/mL; (x) the level of IFN-β that is about: 0 pg/mL to 910 pg/mL, 230 pg/mL to 680 pg/mL, 230 pg/mL to 910 pg/mL, or 0 pg/mL to 680 pg/mL; or (xi) the level of IFN-γ that is about: 55 pg/mL to 110 pg/mL, 70 pg/mL to 95 pg/mL, 70 pg/mL to 110 pg/mL, 55 pg/mL to 95 pg/mL. In some embodiments, the diagnosing is based upon 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 of the one or more measurements.

[00241] Disclosed herein are methods of diagnosing taste or smell disorders in a subject, the methods comprising: (a) obtaining one or more biological samples from the subject; (b) measuring a level of one or more biological substances in the one or more biological samples, wherein the one or more biological substances comprise a cytokine, IgE protein, and/or eosinophils; and (c) comparing the level of the one or more biological substances to a threshold level, thereby diagnosing the hyposmia, anosmia, hypogeusia or ageusia in the subject.

[00242] Also disclosed are methods of diagnosing hyposmia, anosmia, hypogeusia or ageusia in a subject, the methods comprising: (a) obtaining one or more biological samples from the subject; (b) measuring a level of one or biological substances in the one or more biological samples, wherein the one or more biological substances comprise a cytokine, IgE protein, and/or eosinophils; and wherein the measuring comprises, for each biological substance, individually, the use of one or more techniques selected from the following: PCR, Fluorescence Microscopy, Immunoassay, EMIT (Enzyme Multiplied Immunoassay Technique, ELISA (Enzyme Linked Immunosorbent Assay), Radioimmunoassys, Fluorescence Immunoassays, Nuclear magnetic resonanace (NMR), X-ray crystallography, Mass Spectrometry, UV-Vi, Liquid Chromatography (LC)s, Electrophoresis, Arrays, proteomics and protein library screens; and (c) comparing the level of the one or more biological substances to a threshold level, for each bioogical substance individually, from a control population, or each other, thereby diagnosing the hyposmia, anosmia, hypogeusia or ageusia in the subject.

[00243] Also disclosed are methods for identifying a subject who is a member of an orphan drug population, the methods comprising: (a) meausuring a level of one or more biological substances associated with hyposmia, anosmia, hypogeusia or ageusia in one or more biological samples from the subject, wherein the one or more biological substances comprise a cytokine that is IL- la, IL-Ιβ, IL-lra, IL-2, IL-6, IL-10, IL-18, TNF-a, IFN-β, or IFN-γ; a cytokine receptor that is IL-1 RII or IL-2R; IgE protein; eosinophils; a cyclic nucleotide that is cAMP or cGMP; nitric oxide or combinations thereof; (b) comparing, for each of the one or more biological substances individually, the level of the biological substance to a threshold level that is an average level determined in a control population comprising subjects with normal taste and smell function, thereby identifying the subject as a member of the orphan drug population based on the level of the one or more biological substances.

[00244] In some embodiments, the method further comprising evaluating the subject's olfactory function by determining a detection threshold (DT) score, a recognition threshold (RT) score, a magnitude estimation (ME) score, or a combination thereof with a forced-choice, three-stimuli, stepwise-staircase technique using one or more olfaction testing compounds. In some

embodiments, the one or more olfaction testing compounds comprise pyridine, nitrobenzene, thiophene, amyl acetate, or a combination thereof.

[00245] In some embodiments, at least one of the one or more biological samples is a blood sample, a plasma sample, a urine sample, a saliva sample, or a nasal mucus sample. In some embodiments, at least one of the one or more biological samples is a blood sample. In some embodiments, at least one of the one or more biological samples is a nasal mucus sample.

[00246] In some embodiments, the threshold level is based upon an average level in a population with a normal olfactory or taste function.

[00247] In some embodiments, the measuring comprises PCR, Fluorescence Microscopy, Immunoassay, EMIT (Enzyme Multiplied Immunoassay Technique, ELISA (Enzyme Linked Immunosorbent Assay), Radioimmunoassys, Fluorescence Immunoassays, Nuclear magnetic resonanace (NMR), X-ray crystallography, Mass Spectrometry, UV-Vi, Liquid Chromatography (LC)s, Electrophoresis, Arrays, proteomics or protein library screens.

[00248] In some embodiments, the one or more biological substances comprise the cytokine that is IL-la, IL-Ιβ, IL-lra, IL-2, IL-6, IL-10, IL-18, TNF-a, IFN-β, IFN-γ, or a combination thereof. In some embodiments, the cytokine is IL-6. In some embodiments, the one or more biological substances comprise the cytokine receptor that is IL-1 RII, IL-2R, or a combination thereof.

[00249] In some embodiments, the one or more biological substances comprise the cytokine that is IL-6. In some embodiments, the biological sample is a nasal mucus sample. In some embodiments, the threshold level is from about 4 pg/mL to about 12 pg/mL and higher levels indicate that the subject has hyposmia, anosmia, hypogeusia or ageusia.

[00250] In some embodiments, the measuring comprises the use of an antibody. In some embodiments, the antibody is conjugated to an enzyme, a fluorescent molecule, or is radiolabeled. In some embodiments, the measuring comprises an Enzyme-Linked Immunosorbent Assay (ELISA), a sandwich ELISA, a competitive ELISA, a Proximity Ligation Assay (PLA), a western blot, or an immunoprecipitation.

[00251] In some embodiments, the one or more biological substances comprise the IgE protein. In some embodiments, the biological sample is a blood sample. In some embodiments, the threshold level is about: 75 kU/L, 100 kU/L, or 125 kU/L, and higher levels indicate that the subject has hyposmia, anosmia, hypogeusia or ageusia. In some embodiments, the threshold level is about 100 kU/L and higher levels indicate that the subject has hyposmia, anosmia, hypogeusia or ageusia. In some embodiments, the measuring comprises a fluorescence polarization assay.

[00252] In some embodiments, the one or more biological substances comprise the eosiniophils. In some embodiments, the biological sample is a blood sample. In some embodiments, the threshold level is about: 300 cells/HPF (high power field), 350 cells/HPF, or 400 cells/HPF, and higher levels indicate that the subject has hyposmia, anosmia, hypogeusia or ageusia. In some embodiments, the threshold level is about 300 cells/HPF and higher levels indicate that the subject has hyposmia, anosmia, hypogeusia or ageusia. In some embodiments, the measuring is performed with a Coulter counter.

[00253] In some embodiments, the one or more biological substances further comprise cAMP, cGMP, nitric oxide, or a combination thereof.

[00254] Some embodiments further comprise treating the subject for hyposmia, anosmia, hypogeusia or ageusia, wherein the treating comprises administering a pharmaceutical composition comprising an effective amount of one or more phosphodiesterase inhibitors to the subject.

[00255] In some embodiments, the subject is a subject in need of treatment thereof. In some embodiments, the method further comprising treating the subject or the subject in need thereof. [00256] In some embodiments, the pharmaceutical composition is administered orally. In some embodiments, the pharmaceutical composition is administered intranasally. In some

embodiments, the pharmaceutical composition further comprises methylparaben, propylparaben, or a combination thereof.

[00257] In some embodiments, the pharmaceutical composition comprises one or more PDE inhibitors. In some embodiments, the one or more PDE inhibitors comprise a non-selective PDE inhibitor, a PDE-1 selective inhibitor, a PDE-2 selective inhibitor, a PDE-3 selective inhibitor, a PDE-4 selective inhibitor, a PDE-5 selective inhibitor, or a combination thereof. In some embodiments, the one or more PDE inhibitors comprise a non-selective PDE inhibitor that is a methylxanthine derivative. In some embodiments, the methylxanthine derivative is caffeine, theophylline, IBMX (3 -isobutyl-1 -methylxanthine) aminophylline, doxophylline, cipamphylline, neuphylline, pentoxiphylline, or diprophylline. In some embodiments, the one or more PDE inhibitors comprise theophylline.

[00258] In some embodiments, the pharmaceutical composition comprises, or further comprises, an effective amount of an adenylyl cyclase activator, a guanylyl cyclase activator, a cAMP analog, a cGMP analog, or a combination thereof. In some embodiments, the pharmaceutical composition comprises the adenylyl cyclase activator that is forskolin; 1,9-Dideoxyforskolin; 6- [3-(dimethylamino)propionyl]forskolin; adenylyl cyclase toxin; NB001 ; NKH 477; Pituitary adenylate cyclase activating polypeptide-38; Pituitary adenylate cyclase activating polypeptide- 27; or a combination thereof. In some embodiments, the pharmaceutical composition comprises the guanylyl cyclase activator that is A-50619 hydrochloride; atriopeptin II; 6P-Hydroxy-8,13- epoxy-labd-14-en-l 1-one; 9a-Hydroxy-8,13-epoxy-labd-14-en-l 1-one; isoliquiritigenin;

protoporphyrin IX; YC-1; BAY41-2272; CMF-1571; A-350619; BAY 41-8543; BAY 63-2521; BAY58-2667; HMR1766; S3448; or a combination thereof. In some embodiments, the pharmaceutical composition further comprises a steroid. In some embodiments, the

pharmaceutical composition does not comprise a steroid.

[00259] In some embodiments, the pharmaceutical composition comprises, or further comprises, a vasoactive agent that is a potassium channel activator, a calcium blocker, a beta-blocker, an alpha-adrenergic receptor antagonist, a dopamine agonist, an opioid antagonist, a prostaglandin, an endothelin antagonist, or a combination thereof.

[00260] In some embodiments, the pharmaceutical composition comprises, or further comprises, an effective amount of an inhibitor of a pro -inflammatory cytokine. In some embodiments, the inhibitor is an antibody, an antibody fragment, or an antibody mimetic. In some embodiments, the antibody, antibody fragment, or antibody mimetic binds to the pro -inflammatory cytokine. In some embodiments, the antibody, antibody fragment, or antibody mimetic binds to a receptor for the pro -inflammatory cytokine. In some embodiments, the pro-inflammatory cytokine is IL-l , IL-Ιβ, IL-6, IL-18, or TNF-a.

[00261] In some embodiments, the pro-inflammatory cytokine is IL-6. In some embodiments, the inhibitor is an antibody, antibody fragment, or antibody mimetic. In some embodiments, the antibody, antibody fragment, or antibody mimetic binds to IL-6. In some embodiments, the antibody, antibody fragment, or antibody mimetic binds to a receptor for IL-6. In some embodiments, the antibody, antibody fragment, or antibody mimetic is tociluzumab, sarilumab, elsilimomab, siltuximab, sirukumab, BMS-945429, CDP6038, VX30, ARGX-109, or FM101. In some embodiments, the inhibitor is lunasin.

[00262] In some embodiments, the pro-inflammatory cytokine is IL-l . In some embodiments, the inhibitor is an antibody, antibody fragment, or antibody mimetic. In some embodiments, the antibody, antibody fragment, or antibody mimetic binds to IL-l . In some embodiments, the antibody, antibody fragment, or antibody mimetic binds to a receptor for IL-la. In some embodiments, the inhibitor is IL-1RA.

[00263] In some embodiments, the pro-inflammatory cytokine is IL-Ιβ. In some embodiments, the inhibitor is an antibody, antibody fragment, or antibody mimetic. In some embodiments, the antibody, antibody fragment, or antibody mimetic binds to IL-Ιβ. In some embodiments, the antibody, antibody fragment, or antibody mimetic binds to a receptor for IL-Ιβ. In some embodiments, the antibody, antibody fragment, or antibody mimetic is canakinumab.

[00264] In some embodiments, the pro-inflammatory cytokine is TNF-a. In some embodiments, the inhibitor is an antibody, antibody fragment, or antibody mimetic. In some embodiments, the antibody, antibody fragment, or antibody mimetic binds to TNF-a. In some embodiments, the antibody, antibody fragment, or antibody mimetic binds to a receptor for TNF-a. In some embodiments, the antibody, antibody fragment, or antibody mimetic is infliximab, adalimumab, certolizumab pegol, or golimumab. In some embodiments, the inhibitor is etanercept, a xanthine derivative, bupropion, or a 5-HT2A agonist. In some embodiments, the inhibitor is the xanthine derivative that is pentoxifylline. In some embodiments, the inhibitor is the 5-HT2A agonist that is (R)-DOI (2,5-dimethoxy-4-iodoamphetamine), TCB-2 (l-[(7R)-3-bromo-2,5- dimethoxybicyclo[4.2.0]octa-l,3,5-trien-7-yl]methanamine), LSD (lysergic acid diethylamide), or LSZ (Lysergic acid 2,4-dimethylazetidide).

[00265] In some embodiments, the subject experiences a decrease in an odor detection threshold (DT) score or a recognition threshold (RT) score as measured with a forced-choice, three-stimuli, stepwise-staircase technique using one or more olfaction testing compounds after administering the pharmaceutical composition to the subject. In some embodiments, the one or more olfaction testing compounds comprise pyridine, nitrobenzene, thiophene, amyl acetate, or a combination thereof.

[00266] In some embodiments, the subject experiences an increase in a magnitude estimation (ME) score as measured with a forced-choice, three-stimuli, stepwise-staircase technique using one or more olfaction testing compounds after administering the pharmaceutical composition to the subject. In some embodiments, the one or more olfaction testing compounds comprise pyridine, nitrobenzene, thiophene, amyl acetate, or a combination thereof.

[00267] Some embodiments further comprise evaluating the subject's olfactory function by determining a detection threshold (DT) score, a recognition threshold (RT) score, a magnitude estimation (ME) score, or a combination thereof with a forced-choice, three-stimuli, stepwise- staircase technique using one or more olfaction testing compounds.

[00268] In some embodiments, the subject in need thereof experiences a decrease in phantosmia parosmia, phantageusia, or parageusia following administration of the inhibitor.

[00269] In some embodiments, hyposmia, anosmia, hypoguesmia, or aguesima is diagnosed when at least one of the following is ascertained: an eosinophile level measured in at least one of the one or more biological samples is higher than an average level in a control population comprising subjects with normal taste and smell function, an IgE level measured in at least one of the one or more biological samples is higher than an average level in a control population comprising subjects with normal taste and smell function, an IL-6 level measured in at least one of the one or more biological samples is higher than an average level in a control population comprising subjects with normal taste and smell function, a cyclic nucleotide level measured in at least one of the one or more biological samples is higher than an average level in a control population comprising subjects with normal taste and smell function, and/or a NO level measured in at least one of the one or more biological samples is higher than an average level in a control population comprising subjects with normal taste and smell function.

[00270] Fig. I illustrates an exemplary practice of the diagnostic methods disclosed herein. A sample is collected from a subject, as illustrated by a syringe representing an means disclosed herein for the col lection of a biological sample. The method of col lecting the biological sample will depend upon the type of biological sample collected. The biological sample can be analyzed to measure a level of one or more biomarkers from the biological sample using a microscope, or any other means to measure the biomarker level. The levels for each of the one or more biomarkers can be used in a computer implemented diagnosis. The resulting diagnosis based on the biomarker analysis can be sent to a party via a communication media, represented by the computer, diagonal pointing arrow, and printer. Based on the results of the diagnosis, the patient can be treated for a taste or smeli disorder.

METHODS OF TREATMENT

[00271] The substances secreted into saliva and nasal mucus act on local oral and nasal tissues, respectively, to induce physiological effects. There are several effects of gland secretion at distant sites: (1) endocrine-secretions from a gland and subsequent action at a distant site, the secretion carried in blood to the distant site; (2) paracrine-secreted substances act at a distant site within the local reach of the fluid; (3) exocrine-secretions from a gland which have direct local effects, e.g, β-cells in the pancreas which act directly to secrete insulin in response to local changes in blood glucose. This is a one directional effect, a secretion from the gland, into the biological fluid, acting at a distant but local site.

[00272] There are feedback mechanisms such that whatever effects the gland secretion had on its receptor, the receptor also interacted with the site of secretion. For example, increased glucose induces increased secretion of insulin but as insulin secretion increases, insulin receptor number in liver and pancreas change in response to the increased insulin secretion. This feedback concept can also be exemplified by brain secretion of peptide hormones which acted as master feedback mechanisms to control peripheral hormone secretion. Thus, there are interactions between brain, gland and a receptor with the interactions proceeding in both directions. For example, TRH secreted from the brain hypothalamus stimulates pituitary TSH which acts to stimulate thyroid T3 and T₄ which can act back on both pituitary and brain in the form of both long (to brain) and short (to pituitary) feedback loops.

[00273] Henkin, R.I., Olfaction and Taste XI, (Kurihara, K., Suzuki, N., Ogawa, H., Eds.), Springer Verlag, 1994, pp. 568-573, incorporated herein by reference in its entirety, described the concept involving saliva and nasal mucus secretions related to taste and smell function. These results suggest that tastants and odorants affect brain function and vice versa. Since saliva and nasal mucus are the critical factors in maintaining the taste and smell systems, respectively, it is understandable that substances in these fluids also affect brain function and vice versa. Therefore, nasal administration of substances can affect brain function and thereby affect various physiological and pathological problems. For example, nasal administration of leptin (to control obesity), agouti-related protein (to increase appetite in anorexic patients), glucose, albumin, insulin (to treat diabetes), hormones (hormonal disorders), etc. [00274] These effects may act through the large arteriovenous plexus of blood vessels in the nose such that absorption of the substances may be enhanced by direct contact and absorption through these exposed vessels.

Treatment after diagnosis

[00275] Any of the diagnostic methods disclosed herein can further comprise treating the taste or smell disorder in the subject diagnosed with the taste or smell disorder. In some embodiments, the subject is a subject in need thereof.

[00276] In some embodiments, treating comprises administering a pharmaceutical composition to the subject. In some embodiments, the pharmaceutical composition is administered orally. In some embodiments, the pharmaceutical composition is administered intranasally.

[00277] The pharmaceutical composition can comprise an effective amount of one or more phosphodiesterase inhibitors. In some embodiments, the one or more PDE inhibitors comprise a non-selective PDE inhibitor, a PDE-1 selective inhibitor, a PDE-2 selective inhibitor, a PDE-3 selective inhibitor, a PDE-4 selective inhibitor, a PDE-5 selective inhibitor, or a combination thereof. In some embodiments, the one or more PDE inhibitors comprise the non-selective PDE inhibitor that is a methylxanthine derivative. In some embodiments, the methylxanthine derivative is caffeine, theophylline, IBMX (3 -isobutyl-1 -methylxanthine) aminophylline, doxophylline, cipamphylline, neuphylline, pentoxiphylline, or diprophylline. In some

[00278] The pharmaceutical composition can comprise an effective amount of one or more antiinflammatory cytokines. In some embodiments, the one or more anti-inflammatory cytokines comprise IL-lra, IL-10, IFN-γ, or a combination thereof. In some embodiments, the

pharmaceutical composition comprises an effective amount of an antibody, antibody fragment, or antibody mimetic that inhibits a pro -inflammatory cytokine. In some embodiments, the antibody, antibody fragment, or antibody mimetic binds to the pro -inflammatory cytokines. In some embodiments, the antibody, antibody fragment, or antibody mimetic binds to a receptor the pro-inflammatory cytokines. In some embodiments, the pharmaceutical composition comprises the antibody that is a monoclonal antibody. In some embodiments, the monoclonal antibody is a recombinant antibody, a chimeric antibody, a human monoclonal antibody, or a humanized monoclonal antibody. In some embodiments, the pharmaceutical composition comprises the antibody fragment that is a FAB fragment, a FAB2 fragment, a Fv fragment, a ScFv fragment, an antibody light chain, or an antibody heavy chain. In some embodiments, the pharmaceutical composition comprises the antibody mimetic that is an affibody molecule, an affilin, an affitin, an anticalins, an avimers, a DARPins, a fynomer, a Kunitz domain peptide, or a monobody.

[00279] The pharmaceutical composition can comprise an effective amount of an adenylyl cyclase activator, a guanylyl cyclase activator, a cAMP analog, a cGMP analog, or a

[00280] In some embodiments, the pharmaceutical composition further comprises a steroid. In some embodiments, the pharmaceutical composition does not comprise a steroid.

[00281] In some embodiments, the pharmaceutical composition further comprises a vasoactive agent that is a potassium channel activator, a calcium blocker, a beta-blocker, an alpha- adrenergic receptor antagonist, a dopamine agonist, an opioid antagonist, a prostaglandin, an endothelin antagonist, or a combination thereof.

[00282] Treatment efficacy can be demonstrated in a number of ways. In some embodiments, the subject experiences a decrease in a detection threshold (DT) score or a recognition threshold (RT) score as measured with a forced-choice, three-stimuli, stepwise-staircase technique using one or more tastants or odorants after treatment. In some embodiments, the subject experiences an increase in a magnitude estimation (ME) score as measured with a forced-choice, three- stimuli, stepwise-staircase technique using one or more tastants or odorants after treatment. In some embodiments, pro -inflammatory cytokine levels in the subject are lower after treatment. In some embodiments, eosinophil levels in the subject are lower after treatment. In some embodiments, IgE levels in the subject are lower after treatment.

Treatment with nasal mucus or saliva transplantation

[00283] Nasal mucus from persons with normal smell function can contain factors that promote the normal functioning of the olfactory system. Such factors can include growth factors, cytokines, beneficial microorganisms {e.g., bacteria, yeast, etc.). Accordingly, in another aspect, disclosed herein are methods of treating hyposmia or anosmia in a subject in need thereof, the methods comprising: transplanting a nasal mucus sample from a subject with normal olfactory function into the nasal cavity of the subject in need thereof, thereby treating hyposmia or anosmia. In some embodiments, the nasal mucus sample has a volume of about: 1-8 mL, 2-6 mL, 3-5 mL, 4 mL, 1 mL, 500 μί, 100 μί, or 50 μί. Some embodiments further comprise performing a nasal lavage on the subject in need thereof prior to transplanting the nasal mucus sample.

[00284] In some embodiments, the nasal mucus sample is tested for one or more pathogenic organisms prior to transplantation.

[00285] In some embodiments, the nasal mucus sample is obtained from a relative {e.g., an aunt, uncle, sibling, mother, father, son, daughter, cousin, grandparent, grandson, etc.) of the subject in need thereof. In some embodiments, the nasal mucus sample is from the subject in need thereof, wherein the saliva sample was obtained during an asymptomatic period.

[00286] Some embodiments further comprise evaluating the subject in need thereof s olfactory function by determining a detection threshold (DT) score, a recognition threshold (RT) score, a magnitude estimation (ME) score, or a combination thereof with a forced-choice, three-stimuli, stepwise-staircase technique using one or more olfaction testing compounds. In some

[00287] In some embodiments, the nasal mucus transplant is repeated two or more times over a period of time. In some embodiments, the period of time is about: 20 years, 15 years, 10 years, 5 years, 1-365 days, 1-120 days, 1-90 days, 1-60 days, or 1-30 days. In some embodiments, the nasal mucus transplant is repeated about: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more times.

[00288] Some embodiments further comprise at least partially sterilizing the nasal mucus sample prior to transplanting into the nasal cavity of the subject in need thereof. In some embodiments, the at least partially sterilizing comprises filtering the nasal mucus sample. A filter used to partially sterilize the nasal mucus sample can have a pore size size of about 0.005 μΜ, 0.01 μΜ, 0.02 μΜ, 0.03 μΜ, 0.04 μΜ, 0.05 μΜ, 0.06 μΜ, 0.07 μΜ, 0.08 μΜ, 0.09 μΜ, 0.1 μΜ, 0.2 μΜ, 0.3 μΜ, 0.4 μΜ, 0.5 μΜ, 0.6 μΜ, 0.7 μΜ, 0.8 μΜ, 0.9 μΜ, or 1 μΜ. In some embodiments, the filtrate is used in the transplant procedure. In some embodiments, the retentate is used in the transplant procedure. In some embodiments, a retentate from a first filtration is combined with a filtrate from a second filtration and used in the transplant procedure. For example, the first filtration can be with a filter with a pore size to retain microorganisms (e.g., about 0.2 μΜ) and the second filtration can be with a pore size to remove viruses (e.g., about 0.01 μΜ, 0.02 μΜ, 0.03 μΜ, 0.04 μΜ, or 0.05 μΜ).

[00289] In some embodiments, the method does not comprise at least partially sterilizing the nasal mucus sample prior to transplanting into the nasal cavity of the subject in need thereof.

[00290] Some embodiments further comprise administering a steroid-free pharmaceutical dosage unit for intranasal administration comprising a positive amount of one or more PDE inhibitors effective for treating anosmia or hyposmia to the subject in need thereof. In some embodiments, the steroid- fee pharmaceutical dosage unit comprises the positive amount that is less than 45 mg, 30 mg, 15 mg, 10 mg, 5 mg, 1 mg, 500 μg, 250 μg, 120 μg, 80 μg, 40 μg, or 20 μg individually of the one or more PDE inhibitors and a pharmaceutically acceptable carrier.

[00291] In some embodiments, the one or more PDE inhibitors comprise a non-selective PDE inhibitor, a PDE-1 selective inhibitor, a PDE-2 selective inhibitor, a PDE-3 selective inhibitor, a PDE-4 selective inhibitor, a PDE-5 selective inhibitor, a PDE- 10 selective inhibitor, or a combination thereof. In some embodiments, the one or more PDE inhibitors comprise a nonselective PDE inhibitor that is a methylxanthine derivative. In some embodiments, the methylxanthine derivative is caffeine, theophylline, IBMX (3 -isobutyl-1 -methylxanthine) aminophylline, doxophylline, cipamphylline, neuphylline, pentoxiphylline, or diprophylline. In some embodiments, the methylxanthine derivative is theophylline.

[00292] Some embodiments comprise the PDE 1 inhibitor that is vinpocetine. Some

embodiments comprise the PDE 2 inhibitor that is EFINA. Some embodiments comprise the PDE 3 inhibitor that is inamrinone, anagrelide, or cilostazol. Some embodiments comprise the PDE 4 inhibitor that is mesembrine, rolipram, ibudilast, piclamilast, luteolin, drotaverine, or roflumilast. Some embodiments comprise the PDE 5 inhibitor that is sildenafil, tadalafil, vardenafil, udenafil, avanafil, or dipyridamole. Some embodiments comprise the PDE 10 inhibitor that is papaverine.

[00293] In some embodiments, the steroid free pharmaceutical dosage unit is mixed with the nasal mucus sample prior to transplanting the nasal mucus sample into the nasal cavity of the subject in need thereof. In some embodiments, the steroid free pharmaceutical dosage unit is not mixed with the nasal mucus sample prior to transplanting the nasal mucus sample into the nasal cavity of the subject in need thereof.

[00294] In some embodiments, the subject in need thereof was diagnosed with hyposmia or anosmia prior to performing the nasal mucus transplant according to any method disclosed herein.

[00295] Saliva from persons with normal taste function can contain factors that promote the normal functioning of the taste system. Such factors can include growth factors, cytokines, beneficial microorganisms (e.g., bacteria, yeast, etc.). Accordingly, disclosed herein are methods of treating hypogeusia or ageusia in a subject in need thereof, the methods comprising:

transplanting a saliva sample from a subject with normal taste function into the oral cavity of the subject in need thereof, thereby treating hypogeusia or ageusia. In some embodiments, the saliva sample has a volume of about: 1-8 mL, 2-6 mL, 3-5 mL, 4 mL, 1 mL, 500 μί, 100 μί, or 50 μΐ,.

[00296] In some embodiments, the saliva sample is tested for one or more pathogenic organisms prior to transplantation.

[00297] In some embodiments, the saliva sample is obtained from a relative (e.g., an aunt, uncle, sibling, mother, father, son, daughter, cousin, grandparent, grandson, etc.) of the subject in need thereof. In some embodiments, the saliva sample is from the subject in need thereof, wherein the saliva sample was obtained during an asymptomatic period.

[00298] Some embodiments further comprise evaluating the subject in need thereof s taste function by determining a detection threshold (DT) score, a recognition threshold (RT) score, a magnitude estimation (ME) score, or a combination thereof with a forced-choice, three-stimuli, stepwise-staircase technique using one or more tastants. In some embodiments, the one or more tastants comprise sodium chloride (NaCl), sucrose, hydrogen chloride (HC1), urea, or a combination thereof. In some embodiments, the subject in need thereof s taste function is evaluated before and after transplanting the saliva sample.

[00299] In any embodiment disclosed herein, the methods of saliva transplanting can exclude direct transfer of saliva by kissing or other direct mouth-to-mouth contact.

[00300] In some embodiments, the saliva transplant is repeated two or more times over a period of time. In some embodiments, the period of time is about: 20 years, 15 years, 10 years, 5 years, 1-365 days, 1-120 days, 1-90 days, 1-60 days, or 1-30 days. In some embodiments, the saliva transplant repeated about: 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more times. [00301] Some embodiments further comprise at least partially sterilizing the saliva sample prior to transplanting into the oral cavity of the subject in need thereof. In some embodiments, the at least partially sterilizing comprises filtering the saliva sample. A filter used to partially sterilize the saliva sample can have a pore size size of about 0.005 μΜ, 0.01 μΜ, 0.02 μΜ, 0.03 μΜ, 0.04 μΜ, 0.05 μΜ, 0.06 μΜ, 0.07 μΜ, 0.08 μΜ, 0.09 μΜ, 0.1 μΜ, 0.2 μΜ, 0.3 μΜ, 0.4 μΜ, 0.5 μΜ, 0.6 μΜ, 0.7 μΜ, 0.8 μΜ, 0.9 μΜ, or 1 μΜ. In some embodiments, the filtrate is used in the transplant procedure. In some embodiments, the retentate is used in the transplant procedure. In some embodiments, a retentate from a first filtration is combined with a filtrate from a second filtration and used in the transplant procedure. For example, the first filtration can be with a filter with a pore size to retain microorganisms (e.g., about 0.2 μΜ) and the second filtration can be with a pore size to remove viruses (e.g., about 0.01 μΜ, 0.02 μΜ, 0.03 μΜ, 0.04 μΜ, or 0.05 μΜ).

[00302] In some embodiments, the method does not comprise at least partially sterilizing the saliva sample prior to transplanting into the oral cavity of the subject in need thereof.

[00303] Some embodiments further comprise administering a steroid-free pharmaceutical dosage unit for intranasal administration comprising a positive amount of one or more PDE inhibitors effective for treating hypogeusia or ageusia to the subject in need thereof. In some embodiments, the steroid- fee pharmaceutical dosage unit comprises the positive amount that is less than 45 mg, 30 mg, 15 mg, 10 mg, 5 mg, 1 mg, 500 μg, 250 μg, 120 μg, 80 μg, 40 μg, or 20 μg individually of the one or more PDE inhibitors and a pharmaceutically acceptable carrier.

[00304] Some embodiments comprise the PDE 1 inhibitor that is vinpocetine. Some

[00305] In some embodiments, the subject was diagnosed with hypogeusia and/or ageudia accrding to any of the diagnostic methods disclosed herein.

Treatment with Drugs

[00306] Phosphodiesterase treatment can restore taste or smell function in some patients with taste or smell disorders. For example, theophylline is a phosphodiesterase (PDE) inhibitor that can restore taste or smell function through PDE inhibition thereby increasing cAMP, a growth factor which stimulates maturation of olfactory epithelial stem cells, cells whose functions are inhibited among patients with taste or smell disorders. PDE inhibitors (e.g., theophylline) may also restore smell function through other mechanisms. One such mechanism may operate through inhibition of excessive apoptosis, a normal process which, if excessively increased, can become pathological and impair cellular anatomy of the olfactory epithelium and cause hyposmia.

[00307] In some cases, treatment with a PDE inhibitor may return taste or smell function to normal in a dose-dependent manner and may be associated with a dose-dependent decrease in TRAIL. Treatment with a drug may demonstrate a dose dependent decrease in TRAIL which may indicate a decrease in the abnormal apoptotic processes. Biochemical and functional improvement in smell function may also occur after treatment with theophylline. Without limiting the scope of the present invention, other drugs are also considered with in the scope of the present invention for the treatment of various diseases. This is one of the examples of the multiple examples of drugs to treat disease in which changes of various substances found in nasal mucus reflect biochemical normalization and functional improvement in the disease process.

[00308] NO levels in nasal mucus may also change following the treatment of patients with smell loss. Treatment of patients with graded increasing doses of theophylline and measurement of both smell function and NO in nasal mucus in patients with hyposmia. After treatment with theophylline in graded doses there may be increases in nasal mucus NO associated with graded increases in smell function. Treatment with drugs that increase smell function to or toward normal, returns smell function to normal. Measurements of various substances in nasal mucus may be used as an index of both human physiology and pathology of various diseases. Its continual measurement during treatment of the disorders may help in monitoring efficacy of therapy. The detection of NO in nasal mucus provides a non invasive method of diagnosing various diseases related to human physiology and pathology. The methods of the present invention include treatment of diseases by modulating the concentrations of NO by use of drugs or agents. The method of treatment is preferably by nasal administration.

[00309] Disclosed herein are steroid-free pharmaceutical dosage units for intranasal

administration comprising an amount of theophylline effective for treating hyposmia, anosmia, hypogeusia, or ageusia for use in the treatment of hyposmia, anosmia, hypogeusia, or ageusia in a human in need thereof, wherein the steroid- free pharmaceutical dosage unit comprises less than 1 mg of the theophylline and a pharmaceutically acceptable carrier; and wherein the human in need thereof is diagnosed with hyposmia, anosmia, hypogeusia, or ageusia by: (a) obtaining one or more biological samples from the subject; (b) measuring a level of one or more proinflammatory cytokines in at least one of the one or more biological samples; and (c) comparing the level of the one or more pro-inflammatory cytokines to a threshold level, wherein the subject is diagnosed with hyposmia, anosmia, hypogeusia, or ageusia when the level of the one or more pro-inflammatory cytokines is higher than the threshold level.

[00310] Numerous PDEs are known with notable PDEs, their inhibitors and uses for these inhibitors listed below. "Phosphodiesterase inhibitor" or "PDE inhibitor" can refer to any compound that inhibits a phosphodiesterase enzyme, isozyme or allozyme. The term can refer to selective or non-selective inhibitors of cyclic guanosine 3',5'-monophosphate phosphodiesterases (cGMP-PDE) and/or cyclic adenosine 3',5'-monophosphate phosphodiesterases (cAMP-PDE). In some cases, a PDE- 10 inhibitor may be used. In some cases, a PDE-1 inhibitor may be used. In some cases, a PDE-2 selective inhibitor may be used. In some cases a PDE-3 selective inhibitor may be used. In some cases a PDE-5 selective inhibitor may be used. In some cases a combination of one or more PDE inhibitors, as described herein, may be used.

[00311] Theophylline and papaverine are representative members of non-specific PDE inhibitors that can be prescribed orally to treat asthma and chronic obstructive pulmonary disease (COPD) through the relaxation of smooth muscle in the airways. Theophylline has anti-inflammatory effects on the airways that can be useful to combat the abnormal inflammation seen in asthmatics. Most importantly, this anti-inflammatory effect can be obtained at levels in the blood well below that which causes the common side effects seen in most people. Patients with emphysema and chronic bronchitis can also be helped with theophylline when their symptoms are partially related to reversible airway narrowing.

[00312] Theophylline is a methylxanthine derivative; other non-selective phosphodiesterase inhibitors in this class can include caffeine, IBMX (3-isobutyl-l -methylxanthine, aminophylline, doxophylline, cipamphylline, theobromine, pentoxifylline (oxpentifylline) and diprophylline.

[00313] PDE1 selective inhibitors formerly known as calcium- and calmodulin-dependent phosphodiesterases can include eburnamenine-14-carboxylic acid ethyl ester (vinpocetine), which can be used to induce vasorelaxation on cerebral smooth muscle tissue.

[00314] PDE2 decreases aldosterone secretion and can play an important role in the regulation of elevated intracellular concentrations of cAMP and cGMP in platelets. Several regions of the brain can express PDE2 and rat experiments indicate that inhibition of PDE2 enhances memory. PDE2 may play a role in regulation of fluid and cell extravasation during inflammatory conditions as PDE2 is localized to microvessels, especially venous capillary and endothelial cells, but apparently not to larger vessels. PDE2 can also be a pharmacological target for pathological states such as sepsis or in more localized inflammatory responses such as thrombin- induced edema formation in the lung. PDE-2 selective inhibitors can include EHNA (erythro-9- (2-hydroxy-3-nonyl) adenine), 9-(6-phenyl-2-oxohex-3-yl)-2-(3,4-dimethoxybenzyl)-purin-6- one (PDP), and BAY 60-7750.

[00315] The PDE3 family hydro lyzes cAMP and cGMP, but in a manner suggesting that in vivo, the hydrolysis of cAMP is inhibited by cGMP. They also are distinguished by their ability to be activated by several phosphorylation pathways including the PKA and PI3K/PKB pathways. PDE3A is relatively highly expressed in platelets, as well as in cardiac myocytes and oocytes. PDE3B is a major PDE in adipose tissue, liver, and pancreas, as well as in several cardiovascular tissues. Both PDE3A and PDE3B are highly expressed in vascular smooth muscle cells and are likely to modulate contraction.

[00316] PDE3 inhibitors mimic sympathetic stimulation to increase cardiac inotropy,

chronotropy and dromotropy. PDE3 inhibitors can also antagonize platelet aggregation, increase myocardial contractility, and/or enhance vascular and airway smooth muscle relaxation. PDE3A can be a regulator of this process and PDE3 inhibitors can effectively prevent aggregation of platelets. Cilastazol (Pletal), is approved for treatment of intermittent claudication. Without being limited by theory, the mechanism of Cilastazol action is thought to involve inhibition of platelet aggregation along with inhibition of smooth muscle proliferation and vasodilation.

PDE3 -selective inhibitors can include enoximone, milrinone (Primacor), amrinone, cilostamide, cilostazol (Pletal) and trequinsin.

[00317] PDE4 inhibitors can effectively suppress release of inflammatory mediators {e.g., cytokines) and can inhibit the production of reactive oxygen species and immune cell infiltration. PDE4-selective inhibitors can include mesembrine; rolipram; Ibudilast, a neuroprotective and broncho dilator drug used mainly in the treatment of asthma and stroke; and roflumilast (Daxas) and cilomilast (Airflo). PDE4 inhibitors can be effective in treating asthma, arthritis, and psoriasis.

[00318] PDE5s can regulate vascular smooth muscle contraction and can be the molecular target for drugs that can be used to treat erectile dysfunction and/or pulmonary hypertension. In the lung, inhibition of PDE5 can oppose smooth muscle vasoconstriction. PDE5 inhibitors can be used to treat pulmonary hypertension.

[00319] PDE5 -selective inhibitors can include Sildenafil, tadalafil, vardenafil, udenafil and avanafil. [00320] PDE inhibitors can inhibit cellular apoptosis. Without being limited by theory, the mechanism of apoptosis inhibition can include inhibition of TNF alpha, TRAIL and their metabolites. PDE inhibitors can activate the production and secretion of nitric oxide in tissues, which can induce vasorelaxation or vasodilation of blood vessels (e.g., peripheral blood vessels, thereby inhibiting intermittent claudication; the distal extremities; and in the penile region, contributing to penile erection).

[00321] PDE inhibitors useful in the present invention include, for example, filaminast, piclamilast, rolipram, Org 20241 , MCI- 154, roflumilast, toborinone, posicar, lixazinone, zaprinast, sildenafil, pyrazolopyrimidinones (such as those disclosed in WO 98/49166), motapizone, pimobendan, zardaverine, siguazodan, CI-930, EMD 53998, imazodan, saterinone, loprinone hydrochloride, 3-pyridinecarbonitrile derivatives, denbufyllene, albifylline, torbafylline, doxofylline, theophylline, pentoxofylline, nanterinone, cilostazol, cilostamide, MS 857, piroximone, milrinone, aminone, tolafentrine, dipyridamole, papaverine, E4021 , thienopyrimidine derivatives (such as those disclosed in WO 98/17668), triflusal, ICOS-351 , tetrahydropiperazino[l ,2-b]beta-carboline-l ,4-dione derivatives (such as those disclosed in U.S. Pat. No. 5,859,006, WO 97/03985 and WO 97/03675), carboline derivatives, (such as those disclosed in WO 97/43287), 2-pyrazolin-5-one derivatives (such as those disclosed in U.S. Pat. No. 5,869,516), fused pyridazine derivatives (such as those disclosed in U.S. Pat. No.

5,849,741), quinazoline derivatives (such as those disclosed in U.S. Pat. No. 5,614,627), anthranilic acid derivatives (such as those disclosed in U.S. Pat. No. 5,714,993),

imidazo quinazoline derivatives (such as those disclosed in WO 96/26940), and the like. Also included are those phosphodiesterase inhibitors disclosed in WO 99/21562 and WO 99/30697. The disclosures of which are incorporated herein by reference in their entirety. In some embodiments, the intranasal composition does not comprise a PDE5 selective inhibitor.

[00322] Theophylline is an exemplary PDE inhibitor that can be administered according to the methods disclosed herein. In one embodiment, 20 μg/naris of theophylline is administered twice daily. In one embodiment, 40 μg/naris of theophylline is administered once daily. In one embodiment, 40 μg/naris of theophylline is administered twice daily. In one embodiment, 80 μg/naris of theophylline is administered once daily. In one embodiment, 80 μg/naris of theophylline is administered twice daily.

[00323] In some embodiments, the administration of an effective amount of a PDE inhibitor such as theophylline by intranasal administration does not produce a detectable blood level of the PDE inhibitor. In some embodiments, the administration of an effective amount of a PDE inhibitor by intranasal administration produces blood concentration of the PDE inhibitor that are less than 5 mg/dl, 2 mg/ dl, 1 mg/dl, 500 μg/dl, 250 μg/dl, 100 μg/dl, 50 μg/dl, 25 μg/dl, 10 μg/dl, 5 μg/dl, or 1 μg/dl.

[00324] In some embodiments, intranasal administration of an effective amount of a PDE inhibitor such as theophylline increases taste or smell acuity. In some embodiments, the increase in taste or smell acuity is at least 5%, 10%, 20%, 30%, 40%, 50%, 75%, or 100% compared to the untreated state. In other embodiments, taste or smell acuity is increased to at least 5%, 10%, 20%, 30%, 40%, 50%, 75%, or 100% of the acuity of normal individuals. In some

embodiments, taste or smell acuity is measured objectively, while in other embodiments taste or smell acuity is measured subjectively. According to the NIH

(www.nlm.nih.gov/medlinepluse/druginfo/meds/a681006.html) the use of PDE inhibitors such as theophylline can be associated with side effects such as upset stomach, stomach pain, diarrhea, headache, restlessness, insomnia, irritability, vomiting, increased or rapid heart rate, irregular heartbeat, seizures, and/or skin rash. In one embodiment, intranasal administration of PDE inhibitors such as theophylline causes fewer side effects than other routes of administration. In one embodiment, intranasal administration of PDE inhibitors such as theophylline causes less severe side effects than other routes of administration.

[00325] PDE inhibitors such as theophylline can be administered alone or in combination with one or more other active ingredients; for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more other active ingredients, such as any drug disclosed herein.

Treatment with cytokine inhibitors

[00326] In another aspect, disclosed herein are methods of treating taste or smell disorders in a subject in need thereof, the methods comprising administering an effective amount of an inhibitor of a pro -inflammatory cytokine. In some embodiments, the inhibitor is an antibody, an antibody fragment, or an antibody mimetic. In some embodiments, the antibody, antibody fragment, or antibody mimetic binds to the pro-inflammatory cytokine. In some embodiments, the antibody, antibody fragment, or antibody mimetic binds to a receptor for the proinflammatory cytokine. In some embodiments, the pro-inflammatory cytokine is IL-l , IL-Ιβ, IL-6, IL-18, or TNF-a.

[00327] In some embodiments, the pro-inflammatory cytokine is IL-6. In some embodiments, the inhibitor is an antibody, antibody fragment, or antibody mimetic. In some embodiments, the antibody, antibody fragment, or antibody mimetic binds to IL-6. In some embodiments, the antibody, antibody fragment, or antibody mimetic binds to a receptor for IL-6. In some embodiments, the antibody, antibody fragment, or antibody mimetic is tociluzumab, sarilumab, elsilimomab, siltuximab, sirukumab, BMS-945429, CDP6038, VX30, ARGX-109, or FM101. In some embodiments, the inhibitor is lunasin.

[00328] In some embodiments, the pro-inflammatory cytokine is IL-l . In some embodiments, the inhibitor is an antibody, antibody fragment, or antibody mimetic. In some embodiments, the antibody, antibody fragment, or antibody mimetic binds to IL-l . In some embodiments, the antibody, antibody fragment, or antibody mimetic binds to a receptor for IL-l . In some embodiments, the inhibitor is IL-1RA.

[00329] In some embodiments, the pro-inflammatory cytokine is IL-Ιβ. In some embodiments, the inhibitor is an antibody, antibody fragment, or antibody mimetic. In some embodiments, the antibody, antibody fragment, or antibody mimetic binds to IL-Ιβ. In some embodiments, the antibody, antibody fragment, or antibody mimetic binds to a receptor for IL-Ιβ. In some embodiments, the antibody, antibody fragment, or antibody mimetic is canakinumab.

[00330] In some embodiments, the pro-inflammatory cytokine is TNF-a. In some embodiments, the inhibitor is an antibody, antibody fragment, or antibody mimetic. In some embodiments, the antibody, antibody fragment, or antibody mimetic binds to TNF-a. In some embodiments, the antibody, antibody fragment, or antibody mimetic binds to a receptor for TNF-a. In some embodiments, the antibody, antibody fragment, or antibody mimetic is infliximab, adalimumab, certolizumab pegol, or golimumab. In some embodiments, the inhibitor is etanercept, a xanthine derivative, bupropion, or a 5-HT2A agonist. In some embodiments, the inhibitor is the xanthine derivative that is pentoxifylline. In some embodiments, the inhibitor is the 5-HT2A agonist that is (R)-DOI (2,5-dimethoxy-4-iodoamphetamine), TCB-2 (l-[(7R)-3-bromo-2,5- dimethoxybicyclo[4.2.0]octa-l,3,5-trien-7-yl]methanamine), LSD (lysergic acid diethylamide), or LSZ (Lysergic acid 2,4-dimethylazetidide).

[00331] In some embodiments, the subject in need thereof experiences experiences a decrease in an detection threshold (DT) score or a recognition threshold (RT) score for at least one tastant or odorant following administration of the inhibitor. In some embodiments, the subject experiences an increase in a magnitude estimation (ME) score for at least one tastant or odorant following administration of the inhibitor. In some embodiments, the tastant is NaCl, sucrose, HC1, or urea; or the odorant is pyridine, nitrobenzene, thiophene, or amyl acetate. In some embodiments, the subject in need thereof experiences a decrease in phantosmia, parosmia, phantageusia, or parageusia following administration of the inhibitor. In some embodiments, the inhibitor is administered intranasally. [00332] When administered in vivo, the compounds and compositions of the present disclosure can be administered in combination with pharmaceutically acceptable carriers and in dosages described herein. The compounds and compositions of the present disclosure can be formulated as pharmaceutically acceptable neutral (free base), salt forms, or a combination thereof.

Pharmaceutically acceptable salts include, for example, those formed with free amino groups such as those derived from hydrochloric, hydrobromic, hydroiodide, phosphoric, sulfuric, acetic, citric, benzoic, fumaric, glutamic, lactic, malic, maleic, succinic, tartaric, p-toluenesulfonic, methanesulfonic acids, gluconic acid, and the like, and those formed with free carboxyl groups, such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

[00333] The amount of any given active ingredient {e.g., any drug disclosed herein) that will be effective in the prevention or treatment of a particular dysfunction or condition can depend on the nature of the dysfunction or condition, and can be determined by standard clinical techniques, including reference to Goodman and Gilman, supra; The Physician's Desk

Reference, supra; Medical Economics Company, Inc., Oradell, N.J., 1995; and Drug Facts and Comparisons, Inc., St. Louis, Mo., 1993. The precise dose to be used in the formulation can also depend on the route of administration, and the seriousness of the dysfunction or disorder, and can be decided by the physician and the patient's circumstances. In some embodiments, a

therapeutically effective amount of an intranasally administered drug is between about 0.1 and 1000 fold lower than a therapeutically effective amount of the ingredient through another route of administration {e.g., oral administration, intra-muscular injection, subcutaneous injection, intra-venous injection, etc. For example a therapeutically effective amount of an intranasally administered active ingredient can be about 0.1-1000, 0.1-750, 0.1-500, 0.1-250, 0.1-100, 0.1-75, 0.1-50, 0.1-25, 0.1-10, 0.1-1, 1-1000, 1-750, 1-500, 1-250, 1-100, 1-75, 1-50, 1-25, 1-10, 10- 1000, 10-750, 10-500, 10-250, 10-100, 10-75, 10-50, 10-25, 25-1000, 25-750, 25-500, 25-250, 25-100, 25-75, 25-50, 50-1000, 50-750, 50-500, 50-250, 50-100, 50-75, 75-1000, 75-750, 75- 500, 75-250, 75-100, 100-1000, 100-750, 100-500, 100-250, 250-1000, 250-750, 250-500, 500- 1000, 500-750, or 750-1000 fold lower than a therapeutically effective amount of the active ingredient administered by another route.

[00334] When administered intranasally, the amount of an active compound or ingredient can be from about 1 ng to about 1 g. For example, the amount of an active compound or ingredient can be about: 1 ng-1 g, 1 ng-500 mg, 1 ng-250 mg, 1 ng-100 mg, 1 ng-10 mg, 1 ng-1 mg, 1 ng-500 μg, 1 ng-250 μg, 1 ng-100 μg, 1 ng-10 μg, 1 ng-1 μg, 1 ng-500 ng, 1 ng-250 ng, 1 ng-100 ng, 1 ng-10 ng, 10 ng-1 g, 10 ng-500 mg, 10 ng-250 mg, 10 ng-100 mg, 10 ng-10 mg, 10 ng-1 mg, 10 ng-500 μg, 10 ng-250 μg, 10 ng-100 μg, 10 ng-10 μg, 10 ng-1 μg, 10 ng-500 ng, 10 ng-250 ng, 10 ng-100 ng, 100 ng-1 g, 100 ng-500 mg, 100 ng-250 mg, 100 ng-100 mg, 100 ng-10 mg, 100 ng-1 mg, 100 ng-500 μ§, 100 ng-250 μg, 100 ng-100 μ§, 100 ng-10 μg, 100 ng-1 μg, 100 ng- 500 ng, 100 ng-250 ng, 250 ng-1 g, 250 ng-500 mg, 250 ng-250 mg, 250 ng-100 mg, 250 ng-10 mg, 250 ng-1 mg, 250 ng-500 μ§, 250 ng-250 μ§, 250 ng-100 μ§, 250 ng-10 μg, 250 ng-1 μg, 250 ng-500 ng, 500 ng-1 g, 500 ng-500 mg, 500 ng-250 mg, 500 ng-100 mg, 500 ng-10 mg, 500 ng-1 mg, 500 ng-500 μ§, 500 ng-250 μg, 500 ng-100 μ§, 500 ng-10 μg, 500 ng-1 μg, 1 μg-l g, 1 μg-500 mg, 1 μg-250 mg, 1 μg-100 mg, 1 μ§-10 mg, 1 μg-l mg, 1 μg-500 μg, 1 μg-250 μg, 1 μ_§-100 μ_§, 1 μ_§-10 μ_§, 10 μ_§-1 g, 10 μ_§-500 mg, 10 μ_§-250 mg, 10 μ_§-100 mg, 10 μ_§-10 mg, 10 μ_§-1 mg, 10 μ_§-500 μ_§, 10 μ_§-250 μ_§, 10 μ_§-100 μ_§, 100 μ_§-1 g, 100 μ_§-500 mg, 100 μ_§- 250 mg, 100 μg-100 mg, 100 μ§-10 mg, 100 μ -1 mg, 100 μg-500 μ§, 100 μg-250 μ§, 250 μg-l g, 250 μg-500 mg, 250 μg-250 mg, 250 μg-100 mg, 250 μ§-10 mg, 250 μ -1 mg, 250 μg-500 μ§, 500 μ -1 g, 500 μg-500 mg, 500 μg-250 mg, 500 μg-100 mg, 500 μ§-10 mg, 500 μ -1 mg, 1 mg-1 g, 1 mg-500 mg, 1 mg-250 mg, 1 mg-100 mg, 1 mg-10 mg, 10 mg-1 g, 10 mg-500 mg, 10 mg-250 mg, 10 mg-100 mg, 100 mg-1 g, 100 mg-500 mg, 100 mg-250 mg, 250 mg-1 g, 250 mg- 500 mg, or 500 mg-1 g. The inventive embodiments envision that any of the recited ranges can be combined to form the ranges for administration.

[00335] Sources of information for the drugs disclosed herein can include Goodman and Gilman, The Pharmacological Basis of Therapeutics (9th Ed.), McGraw-Hill, Inc. (1995), The Physician's Desk Reference (49th Ed.), Medical Economics (1995), Drug Facts and Comparisons (1993 Ed), Facts and Comparisons (1993), and The Merck Index (12th Ed.), Merck & Co., Inc. (1996), the disclosures of each of which are incorporated herein by reference in their entirety.

[00336] Any of the active compounds or ingredients disclosed herein can be administered as a composition or a pharmaceutical composition. The compositions or pharmaceutical compositions can be a solid or a liquid. A solid composition can be a powder.

[00337] Any of the active compounds or ingredients disclosed herein can be administed together. For example, two or more active compounds or ingredients can be administered in a single composition or separately administered as separate compositions.

[00338] Any composition or pharmaceutical composition containing one or more active compounds or ingredients can contain one or more excipients. The one or more excipients can be any excipient disclosed herein. The one or more excipients can be any excipient disclosed in Rowe, et al. Handbook of Pharmaceutical Excipients (2012, 7th Ed.), Pharmaceutical Press, which is hereby incorporated by reference in its entirety. [00339] A composition or pharmaceutical composition containing one or more active compounds or ingredients disclosed herein can be administered in an amount of from about 10 ng to about 2 g. For example, the composition or pharmaceutical composition containing one or more active compounds or ingredients disclosed herein can be administered in an amount of about: 10 ng-2 g,10 ng-1 g,10 ng-500 mg, 10 ng-250 mg, 10 ng-100 mg, 10 ng-10 mg, 10 ng-1 mg, 10 ng-500 μ_§, 10 ng-250 μ_§, 10 ng-100 μ_§, 10 ng-10 μ_§, 10 ng-1 μ_§, 10 ng-500 ng, 10 ng-250 ng, 10 ng- 100 ng, 100 ng-2 g, 100 ng-1 g, 100 ng-500 mg, 100 ng-250 mg, 100 ng-100 mg, 100 ng-10 mg, 100 ng-1 mg, 100 ng-500 μ& 100 ng-250 μg, 100 ng-100 μg, 100 ng-10 μg, 100 ng-1 μ 100 ng-500 ng, 100 ng-250 ng, 250 ng-2 g, 250 ng-1 g, 250 ng-500 mg, 250 ng-250 mg, 250 ng-100 mg, 250 ng-10 mg, 250 ng-1 mg, 250 ng-500 μg, 250 ng-250 μg, 250 ng-100 μ& 250 ng-10 μg, 250 ng-1 μg, 250 ng-500 ng, 500 ng-2 g, 500 ng-1 g, 500 ng-500 mg, 500 ng-250 mg, 500 ng- 100 mg, 500 ng-10 mg, 500 ng-1 mg, 500 ng-500 μg, 500 ng-250 μg, 500 ng-100 μ& 500 ng-10 μg, 500 ng-1 μg, 1 μg-2 g, 1 μg-l g, 1 μg-500 mg, 1 μg-250 mg, 1 μg-100 mg, 1 μg-10 mg, 1 μg-l mg, 1 μg-500 μg, 1 μg-250 μg, 1 μg-100 μg, 1 μg-10 μg, 10 μg-2 g, 10 μ -1 g, 10 μg-500 mg, 10 μg-250 mg, 10 μg-100 mg, 10 μg-10 mg, 10 μg-l mg, 10 μg-500 μ& 10 μg-250 μ& 10 μg-100 μ& 100 μg-2 g, 100 μg-l g, 100 μg-500 mg, 100 μg-250 mg, 100 μg-100 mg, 100 μg-10 mg, 100 μg-l mg, 100 μg-500 μg, 100 μg-250 μ¾ 250 μg-2 g, 250 μg-l g, 250 μg-500 mg, 250 μg-250 mg, 250 μg-100 mg, 250 μg-10 mg, 250 μg-l mg, 250 μg-500 μg, 500 μg-2 g, 500 μg-l g, 500 μg-500 mg, 500 μg-250 mg, 500 μg-100 mg, 500 μg-10 mg, 500 μg-l mg, 1 mg-2 g, 1 mg-1 g, 1 mg-500 mg, 1 mg-250 mg, 1 mg-100 mg, 1 mg-10 mg, 10 mg-2 g, 10 mg-1 g, 10 mg- 500 mg, 10 mg-250 mg, 10 mg-100 mg, 100 mg-2 g, 100 mg-1 g, 100 mg-500 mg, 100 mg-250 mg, 250 mg-2 g, 250 mg-1 g, 250 mg-500 mg, 500 mg-2 g, 500 mg-1 g, or 1 g-2 g.

[00340] A composition or pharmaceutical composition containing one or more active compounds or ingredients disclosed herein can be administered one or more times per day; for example, 1, 2, 3, 4, 5, 6, or more times per day. The composition or pharmaceutical composition containing one or more active compounds or ingredients disclosed herein can be administered every other day, every third day, every fourth day, every fifth day, or every sixth day. The composition or pharmaceutical composition containing one or more active compounds or ingredients disclosed herein can be administered every week, every two weeks, every three weeks, every month, every two months, every three months, every four months, every five months, or every six months.

[00341] The composition or pharmaceutical composition containing one or more active compounds or ingredients disclosed herein can be administered for any suitable period of time. For example, the composition or pharmaceutical composition containing one or more active compounds or ingredients disclosed herein can be administered for about: 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years, 4 years, 5 years, or longer. The composition or pharmaceutical composition containing one or more active compounds or ingredients disclosed herein can be administered for an indefinite period of time. The composition or pharmaceutical composition containing one or more active compounds or ingredients disclosed herein can be administered indefinitely.

Dosage Forms

[00342] Aerosols

[00343] By "aerosol" is meant any composition of one or more active ingredients or drugs that is administered as an aerosolized formulation, including for example an inhalation spray, inhalation solution, inhalation suspension, a nebulized solution, or nasal spray. Aerosolized formulations can deliver high concentrations of one or more active ingredients or drugs directly to the airways with low systemic absorption. Solutions for aerosolization typically contain at least one therapeutically active ingredient dissolved or suspended in an aqueous solution that may further include one or more excipients {e.g., preservatives, viscosity modifiers, emulsifiers, or buffering agents). The solution acts as a carrier for the one or more active ingredients or drugs. In some embodiments, the preservative is methylparaben or propylparaben. These formulations are intended for delivery to the respiratory airways by inspiration.

[00344] A major limitation of pulmonary delivery is the difficulty of reaching the deep lung. To achieve high concentrations of the active ingredient(s) solution in both the upper and lower respiratory airways, the active ingredient(s) solution can be nebulized in jet nebulizers, a ultrasonic nebulizer, or an electronic nebulizer particularly those modified with the addition of one-way flow valves, such as for example, the Pari LC Plus.TM. nebulizer, commercially available from Pari Respiratory Equipment, Inc., Richmond, Va., which delivers up to 20% more drug than other unmodified nebulizers.

[00345] The pH of the formulation may be any suitable pH. The aerosol may be acidic or basic, The pH of the formulation is may be maintained between 4.5 and 8.0. In some cases, the pH of the aerosol formulation may be at least 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7,

5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9,

8.0, or 9.0. In some cases, the pH of the aerosol formulation may be at most 4.5, 4.6, 4.7, 4.8,

4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0,

7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, or 9.0. [00346] In some cases, the pH of the aerosol may be above pH 8. In some cases, the aerosol may be 8.1 , 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9 or 9.0 in pH.

[00347] Drops and Gels

[00348] In some embodiments, the one or more active ingredients or drugs are directly applied to the nasal or lingual epithelium as a liquid, cream, lotion, ointment or gel. These fluids or semifluids contain at least one therapeutically active ingredient and may further include at least one excipient (e.g., preservatives, viscosity modifiers, emulsifiers, or buffering agents) that are formulated for administration as nose drops, or applied with an applicator to the inside of the nasal passages. In some embodiments, the preservative is methylparaben or propylparaben. The pH of the formulation is preferably maintained between 4.5 and 7.0, more preferably between 5.0 and 7.0 and most preferably between 5.5 and 6.5. The osmolarity of the formulation can also be adjusted to osmolarities of about 250 to 350 mosm/L.

[00349] Dry Powder Formulation

[00350] As an alternative therapy to aerosol, liquid or gel delivery, the one or more active ingredients or drugs can be administered in a dry powder formulation for efficacious delivery into the nasal cavity and/or endobronchial space. Dry powder formulation can be convenient; for example, dry powder formulations can require no further handling by a physician, pharmacist or patient such as diluting or reconstituting the agent as is often required with nebulizers.

Furthermore, dry powder delivery devices can be sufficiently small and can be portable. Dry powder formulations can also be applied directly on the lingual epithelium.

[00351] For dry powder formulations, one or more active ingredients or drugs and/or carrier can be processed to median diameter ranging from about 0.001-250 μιη. Processing of dry powder formulations can comprise media milling, jet milling, spray drying, super-critical fluid energy, or particle precipitation techniques. Particles of a desired size ranges can also be obtained through the use of sieves. Frequently, milled particles are passed through one or more sieves to isolate a desired size range. In some embodiments intended for pulmonary administration, the dry powder formulation has a median diameter ranging from 0.01-25 μιη, 0.1-10 μιη, 1-10 μιη, 1-5 μιη, or 2- 5 μιη. In further embodiments intended for pulmonary administration, the dry powder formulation can have a median diameter ranging less than 20 μιη, 10 μιη, 5 μιη, 4, μιη, 3 μιη, 2 μιη, or 1 μιη. In some embodiments intended for nasal administration, the dry powder formulation can have a median diameter ranging from 1-250 μιη, 5-200 μιη, 10-150 μιη, 10-100 μιη, 10-50 μιη, 15-100 μιη, 15-50 μιη, or 20-60 μιη. In further embodiments intended for nasal administration, the dry powder formulation can have a median diameter of less than 250 μιη, 200 μιη, 150 μιη, 100 μιη, 75 μιη, 60 μιη, 50 μιη, 40 μιη or 30 μιη. In other embodiments intended for nasal administration, the dry powder formulation can have a median diameter of at least 20 μιη, 30 μιη, 40 μιη, 50 μιη, 60 μιη, 75 μιη, 100 μιη, 150 μm or 200 μιη.

[00352] In some embodiments, a pharmaceutically acceptable carrier for the present

compositions and formulations include but are not limited to amino acids, peptides, proteins, non-biological polymers, biological polymers, simple sugars, carbohydrates, gums, inorganic salts and metal compounds which may be present singularly or in combination. In some embodiments, the pharmaceutically acceptable carrier comprises native, derivatized, modified forms, or combinations thereof.

[00353] In some embodiments, useful proteins include, but are not limited to, gelatin or albumin. In some embodiments, useful sugars that can serve as pharmaceutically acceptable carriers include, but are not limited to fructose, galactose, glucose, lactitol, lactose, maltitol, maltose, mannitol, melezitose, myoinositol, palatinite, raffinose, stachyose, sucrose, trehalose, xylitol, hydrates thereof, and combinations of thereof.

[00354] In some embodiments, useful carbohydrates that can serve as pharmaceutically acceptable carriers include, but are not limited to starches such as corn starch, potato starch, amylose, amylopectin, pectin, hydroxypropyl starch, carboxymethyl starch, and cross-linked starch. In other embodiments, useful carbohydrates that can serve as pharmaceutically acceptable carriers include, but are not limited to cellulose, crystalline cellulose, micro crystalline cellulose, a-cellulose, methylcellulose, hydroxypropyl cellulose, carboxymethyl cellulose, ethyl cellulose, hydroxypropyl methyl cellulose, and cellulose acetate.

[00355] In some embodiments, the composition or formulation includes an excipient. Useful excipients include, but are not limited to, fluidizers, lubricants, adhesion agents, surfactants, acidifying agents, alkalizing agents, agents to adjust pH, antimicrobial preservatives,

antioxidants, anti-static agents, buffering agents, chelating agents, humectants, gel-forming agents, or wetting agents. Excipients also include coloring agents, coating agents, sweetening, flavoring and perfuming and other masking agents. The compositions and formulations of this invention may include a therapeutic agent with an individual excipient or with multiple excipients in any suitable combination, with or without a carrier.

[00356] The dry powder formulations of the present invention can be used directly in metered dose or dry powder inhalers. With dry powder inhalers, the inspiratory flow of the patient can accelerate the powder out of the device and into the nasal and/or oral cavity. Alternatively, dry powder inhalers can employ an air source, a gas source, or electrostatics, to deliver the therapeutic agent. The dry powder formulations can be temperature stable and have a

physiologically acceptable pH of about 4.0-7.5, e.g., about 6.5 to 7.0. Effective Dosages

[00357] Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredient is contained in a therapeutically or prophylactically effective amount, i.e., in an amount effective to achieve therapeutic or prophylactic benefit. Of course, the actual amount effective for a particular application will depend, inter alia, on the condition being treated and the route of administration. Determination of an effective amount is well within the capabilities of those skilled in the art, especially in light of the disclosure herein.

[00358] Therapeutically effective amounts for use in humans can be determined from animal models. For example, a dose for humans can be formulated to achieve circulating concentration that has been found to be effective in animals. The amount administered can be the same amount administered to treat a particular disease or can be an amount lower than the amount

administered to treat that particular disease. Patient doses for oral administration of the drug may range from about 1 μg-l gm/day. The dosage may be administered once per day or several or multiple times per day. The amount of the drug administered to practice methods of the present invention will of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration and the judgment of the prescribing physician. The dose used to practice the invention can produce the desired therapeutic or prophylactic effects, without producing serious side effects.

Routes of administration

[00359] The methods of treatment in the invention include by way of example only, oral administration, transmucosal administration, buccal administration, nasal administration such as inhalation, parental administration, intravenous, subcutaneous, intramuscular, sublingual, transdermal administration, and rectal administration.

[00360] In some embodiments of the present invention, the method of treatment is by nasal administration or inhalation. Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders. The liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described supra. The compositions can be administered by the oral or nasal respiratory route for local or systemic effect. Compositions in preferably pharmaceutically acceptable solvents may be nebulized by use of inert gases. Nebulized solutions may be inhaled directly from the nebulizing device or the nebulizing device may be attached to a face mask tent, or intermittent positive pressure breathing machine. Solution, suspension, or powder

compositions may be administered, preferably orally or nasally, from devices that deliver the formulation in an appropriate manner. [00361] In some embodiments of the present invention, the method of treatment is by oral administration. Oral administration can be presented as discrete dosage forms, such as capsules, cachets, or tablets, or liquids or aerosol sprays each containing a predetermined amount of an active ingredient as a powder or in granules, a solution, or a suspension in an aqueous or nonaqueous liquid, an oil-in-water emulsion, or a water-in-oil liquid emulsion. Such dosage forms can be prepared by any of the methods of pharmacy, but all methods include the step of bringing the active ingredient into association with the carrier, which constitutes one or more necessary ingredients. In general, the compositions are prepared by uniformly and intimately admixing the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation. For example, a tablet can be prepared by compression or molding, optionally with one or more accessory ingredients.

Compressed tablets can be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as powder or granules, optionally mixed with an excipient such as, but not limited to, a binder, a lubricant, an inert diluent, and/or a surface active or dispersing agent. Molded tablets can be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

[00362] An active ingredient can be combined in an intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques. The carrier can take a wide variety of forms depending on the form of preparation desired for administration. In preparing the compositions for an oral dosage form, any of the usual pharmaceutical media can be employed as carriers, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, and the like in the case of oral liquid preparations (such as suspensions, solutions, and elixirs) or aerosols; or carriers such as starches, sugars, micro- crystalline cellulose, diluents, granulating agents, lubricants, binders, and disintegrating agents can be used in the case of oral solid preparations, in some embodiments without employing the use of lactose. For example, suitable carriers include powders, capsules, and tablets, with the solid oral preparations. If desired, tablets can be coated by standard aqueous or nonaqueous techniques.

[00363] Examples of suitable fillers for use in the pharmaceutical compositions and dosage forms disclosed herein include, but are not limited to, talc, calcium carbonate (e.g., granules or powder), micro crystalline cellulose, powdered cellulose, dextrates, kaolin, mannitol, silicic acid, sorbitol, starch, pre-gelatinized starch, and mixtures thereof.

[00364] Disintegrants may be used in the method of treatment of the present invention to provide tablets that disintegrate when exposed to an aqueous environment. Disintegrants that can be used to form pharmaceutical compositions and dosage forms of the invention include, but are not limited to, agar-agar, alginic acid, calcium carbonate, micro crystalline cellulose, croscarmellose sodium, crospovidone, polacrilin potassium, sodium starch glycolate, potato or tapioca starch, other starches, pre-gelatinized starch, other starches, clays, other algins, other celluloses, gums or mixtures thereof.

[00365] Lubricants which can be used to form pharmaceutical compositions and dosage forms of the invention include, but are not limited to, calcium stearate, magnesium stearate, mineral oil, light mineral oil, glycerin, sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid, sodium lauryl sulfate, talc, hydrogenated vegetable oil (e.g., peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil), zinc stearate, ethyl oleate, ethyl laureate, agar, or mixtures thereof. Additional lubricants include, for example, a syloid silica gel, a coagulated aerosol of synthetic silica, or mixtures thereof. A lubricant can optionally be added, in an amount of less than about 1 weight percent of the pharmaceutical composition.

[00366] When aqueous suspensions and/or elixirs are desired for oral administration, the essential active ingredient therein may be combined with various sweetening or flavoring agents, coloring matter or dyes and, if so desired, emulsifying and/or suspending agents, together with such diluents as water, ethanol, propylene glycol, glycerin and various combinations thereof.

[00367] The tablets can be uncoated or coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate can be employed. Formulations for oral use can also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example, peanut oil, liquid paraffin or olive oil.

[00368] Surfactant which can be used to form pharmaceutical compositions and dosage forms of the invention include, but are not limited to, hydrophilic surfactants, lipophilic surfactants, and mixtures thereof. That is, a mixture of hydrophilic surfactants may be employed, a mixture of lipophilic surfactants may be employed, or a mixture of at least one hydrophilic surfactant and at least one lipophilic surfactant may be employed. A solubilizer may also be added to increase the solubility of the hydrophilic drug and/or other components, such as surfactants, or to maintain the composition as a stable or homogeneous solution or dispersion.

[00369] Mixtures of solubilizers may be used. Examples include, but not limited to, triacetin, triethylcitrate, ethyl oleate, ethyl caprylate, dimethylacetamide, N-methylpyrrolidone, N- hydroxyethylpyrrolidone, polyvinylpyrrolidone, hydroxypropyl methylcellulose, hydroxypropyl cyclodextrins, ethanol, polyethylene glycol 200-100, glycofurol, transcutol, propylene glycol, and dimethyl isosorbide. Particularly preferred solubilizers include sorbitol, glycerol, triacetin, ethyl alcohol, PEG-400, glycofurol and propylene glycol.

[00370] The compositions for the treatment can further include one or more pharmaceutically acceptable additives and excipients. Such additives and excipients include, without limitation, detackifiers, anti-foaming agents, buffering agents, polymers, antioxidants, preservatives, chelating agents, viscomodulators, tonicifiers, flavorants, colorants, odorants, opacifiers, suspending agents, binders, fillers, plasticizers, lubricants, and mixtures thereof.

[00371] In addition, an acid or a base may be incorporated into the composition to facilitate processing, to enhance stability, or for other reasons. Examples of pharmaceutically acceptable bases include amino acids, amino acid esters, ammonium hydroxide, potassium hydroxide, sodium hydroxide, sodium hydrogen carbonate, aluminum hydroxide, calcium carbonate, magnesium hydroxide, magnesium aluminum silicate, synthetic aluminum silicate, synthetic hydrocalcite, magnesium aluminum hydroxide, diisopropylethylamine, ethanolamine, ethylenediamine, triethanolamine, triethylamine, triisopropanolamine, trimethylamine, tris(hydroxymethyl)aminomethane (TRIS) and the like. Suitable acids are pharmaceutically acceptable organic or inorganic acids. Examples of suitable inorganic acids include hydrochloric acid, hydrobromic acid, hydriodic acid, sulfuric acid, nitric acid, boric acid, phosphoric acid, and the like. Examples of suitable organic acids include acetic acid, acrylic acid, adipic acid, alginic acid, alkanesulfonic acids, amino acids, ascorbic acid, benzoic acid, boric acid, butyric acid, carbonic acid, citric acid, fatty acids, formic acid, fumaric acid, gluconic acid,

hydro quino sulfonic acid, isoascorbic acid, lactic acid, maleic acid, methanesulfonic acid, oxalic acid, para-bromophenylsulfonic acid, propionic acid, p-toluenesulfonic acid, salicylic acid, stearic acid, succinic acid, tannic acid, tartaric acid, thioglycolic acid, toluenesulfonic acid, uric acid and the like.

[00372] The forms in which the compositions of the present invention may be incorporated for administration by injection include aqueous or oil suspensions, or emulsions, with sesame oil, corn oil, cottonseed oil, or peanut oil, as well as elixirs, mannitol, dextrose, or a sterile aqueous solution, and similar pharmaceutical vehicles.

[00373] Aqueous solutions in saline are also conventionally used for injection. Ethanol, glycerol, propylene glycol, liquid polyethylene glycol, and the like (and suitable mixtures thereof), cyclodextrin derivatives, and vegetable oils may also be employed. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.

[00374] The compositions for delivery can be formulated into preparations in solid, semi-solid, or liquid forms suitable for local or topical administration, such as gels, water soluble jellies, creams, lotions, suspensions, foams, powders, slurries, ointments, solutions, oils, pastes, suppositories, sprays, emulsions, saline solutions, dimethylsulfoxide (DMSO)-based solutions. In general, carriers with higher densities are capable of providing an area with a prolonged exposure to the active ingredients. In contrast, a solution formulation may provide more immediate exposure of the active ingredient to the chosen area.

[00375] In some embodiments of the present invention, the method of treatment can be transdermal. Transdermal patches may be used to provide continuous or discontinuous infusion in controlled amounts, either with or without therapeutic agent. The construction and use of transdermal patches for the delivery of pharmaceutical agents is well known in the art. See, e.g., U.S. Pat. Nos. 5,023,252, 4,992,445 and 5,001,139. Such patches may be constructed for continuous, pulsatile, or on demand delivery of pharmaceutical agents.

[00376] Pharmaceutical compositions may also be prepared with one or more pharmaceutically acceptable excipients suitable for sublingual, buccal, rectal, intraosseous, intraocular, intranasal, epidural, or intraspinal administration. Preparations for such pharmaceutical compositions are well-known in the art. See, e.g., See, e.g., Anderson, Philip O.; Knoben, James E.; Troutman, William G, eds., Handbook of Clinical Drug Data, Tenth Edition, McGraw-Hill, 2002; Pratt and Taylor, eds., Principles of Drug Action, Third Edition, Churchill Livingston, New York, 1990; Katzung, ed., Basic and Clinical Pharmacology, Ninth Edition, McGraw Hill, 20037ybg;

Goodman and Gilman, eds., The Pharmacological Basis of Therapeutics, Tenth Edition, McGraw Hill, 2001 ; Remingtons Pharmaceutical Sciences, 20th Ed., Lippincott Williams & Wilkins., 2000; Martindale, The Extra Pharmacopoeia, Thirty-Second Edition (The

Pharmaceutical Press, London, 1999); all of which are incorporated by reference herein in their entirety.

Dosing

[00377] The dose and dosage period of the pharmaceutical composition of the present disclosure is a dose and period sufficient for realizing the intended action, which may be adequately adjusted depending on the administration route, frequency of administration per day, seriousness of the symptoms, body weight, age, and other factors.

[00378] Doses of the aforementioned compositions as the active ingredient can be suitably decided depending on the purpose of administration, i.e., therapeutic or preventive treatment, nature of a disease to be treated or prevented, conditions, body weight, age, sexuality and the like of a patient. The practically desirable method and sequence for administration varies depending on the purpose of administration, i. e., therapeutic or preventive treatment, nature of a disease to be treated or prevented, conditions, body weight, age, sexuality and the like of a patient. The optimum method and sequence for administration of the compounds described in detail herein under preset given conditions may be suitably selected by those skilled in the art with the aid of the routine technique and the information contained in the present specification and field of invention.

[00379] In some cases, administration of the pharmaceutical composition of this disclosure may be administered in terms of a dose or unit dose. A pharmaceutical composition of this disclosure may comprise one or more active agents each in an amount ranging from about 1 ug - 1 g, for example, about 10 ug, about 20 ug, about 30 ug, about 40 mg, about 50 ug, about 60 ug, about 70 ug, about 80 ug, about 90 ug, or about 100 ug. In some cases, a pharmaceutical composition of this disclosure may comprise one or more active agents, each in an amount individually ranging from about 5 ug - 100 mg, or least about: 5 ug, 10 ug, 15 ug, 20 ug, 25 ug, 30 ug, 35 ug, 40 ug, 45 ug, 50 ug, 60 ug, 70 ug, 80 ug, 90 ug, 100 ug, 200 ug, 300 ug, 400 ug, 500 ug, 600 ug, 700 ug, 800 ug, 900 ug, 1 mg, 10 mg, 25 mg, 50 mg, 75 mg or 100 mg.

[00380] In some cases, administration of the pharmaceutical composition of this disclosure may be administered in terms unit dose, such as mg per kg of a subject's body weight. For example, in some cases, a pharmaceutical composition of this disclosure may administer an amount of active agent, in each case individually ranging from about 0.001 mg/kg - 10 mg/kg, for example at least about 0.001 mg/kg, 0.005 mg/kg, 0.01 mg/kg, 0.015 mg/kg, 0.02 mg/kg, 0.025 mg/kg, 0.03 mg/kg, 0.035 mg/kg, 0.040 mg/kg, 0.050 mg/kg, 0.060 mg/kg, 0.070 mg/kg, 0.080 mg/kg, 0.090 mg/kg, 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.6 mg/kg, 0.7 mg/kg, 0.8 mg/kg, 0.9 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 3.0 mg/kg, 4.0 mg/kg, 5.0 mg/kg, 6.0 mg/kg, 7.0 mg/kg, 8.0 mg/kg, 9.0 mg/kg or 10.0 mg/kg.

[00381] In some cases, the entire dose may be administered at once or in several divided doses. In some cases, the pharmaceutical composition of this disclosure may be divided and

administered in 1-100 doses. In some cases, the pharmaceutical composition of this disclosure may be divided and administered in at least 1, 2, 3, 4, 5, ,6 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 doses. In some cases, the pharmaceutical composition of this disclosure may be divided and administered in at most 1, 2, 3, 4, 5, ,6 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 doses.

Dosing Schedule [00382] The pharmaceutical compositions of this disclosure may also be administered according to various dosing schedules. The pharmaceutical compositions of this disclosure may also be administered hourly, twice a day, once a day, one a week, one a month, once every 6 months, 1 a year, or once every 5 years. In some cases the duration of administration of the pharmaceutical compositions of this disclosure may range from 1 day to indefinite administration.

[00383] If indicated by the physician, administration may be started at a dose lower than the recommended dosage at the first day, and then, the dose may be gradually increased to the maximum daily dose as the maintenance dose. The dose may be reduced depending on the conditions of the patient. Lower daily dose is preferable in view of reducing any potential side effects.

[00384] Kits/Articles of Manufacture

[00385] For use of the therapeutic compositions described herein, kits and articles of

manufacture are also described. In some embodiments, such kits include a carrier, package, or container that is compartmentalized to receive one or more blister packs, bottles, tubes, capsules, and the like. In certain embodiments, the pharmaceutical compositions are presented in a pack or dispenser device which contains one or more unit dosage forms containing a compound provided herein. In other embodiments, the pack contains metal or plastic foil, such as a blister pack. In some embodiments, the pack contains capsules, vials, or tubes. In other embodiments, the pack or dispenser device is accompanied by instructions for administration. In some embodiments, the dispenser is disposable or single use, while in other embodiments, the dispenser is reusable. In certain embodiments, the pharmaceutical formulations are preloaded into the device.

[00386] In still other embodiments, the pack or dispenser also accompanied with a notice as required by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals. This notice can state that the drug is approved by the agency for human or veterinary

administration. Such notice, for example, can be the labeling approved by the U.S. Food and Drug Administration for prescription drugs, or the approved product insert. Compositions containing a compound provided herein formulated in a compatible pharmaceutical carrier can also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.

[00387] The articles of manufacture provided herein can also contain an administration or dispensing device. Examples of administration devices include pulmonary inhalers and intranasal applicators. Pumps can be provided with the inhalers and intranasal devices, or the pumps may be built into the devices. Alternatively, a propellant can be included with or it may be stored within the devices.

[00388] Such kits can optionally comprise an identifying description or label for the containers. In further embodiments, the label is on a container with letters, numbers or other characters forming the label and attached, molded or etched into the container itself; a label can be associated with a container when it is present within a receptacle or carrier that also holds the container, e.g., as a package insert. In some embodiments, a label is used to indicate that the contents are to be used for a specific therapeutic application. In yet other embodiments, the label indicates directions for use of the contents, such as in the methods described herein. In some embodiments, a set of instructions can also be included, generally in the form of a package insert. The informational material can contain instructions on how to dispense the

pharmaceutical composition, including description of the type of patients who may be treated, the schedule (e.g., dose and frequency), and the like.

[00389] The present disclosure also relates to a set (kit) comprising separate packs of kits; for example, as assembled for shipping or for patient convenience, such as a weekly, biweekly or monthly supply of a medicament.

[00390] Business Methods

[00391 ] One or more computers may be utilized in the diagnostic methods disclosed herein, such as a computer 800 as illustrated in Fig. 2. The computer 8Θ0 may be used for managing subject and sample information such as sample or subject tracking, database management, analyzing biomarker data, analyzing cytological data, storing data, billing, marketing, reporting results, or storing results. The computer may include a monitor 807 or other graphical interface for displaying data, results, billing information, marketing information (e.g. demographics), subject information, or sample information. The computer may also include means for data or information input 816, 815. The computer may include a processing unit 801 and fixed 803 or removable 811 media or a combination thereof. The computer may be accessed by a user in physical proximity to the computer, for example via a keyboard and/or mouse, or by a user 822 that does not necessarily have access to the physical computer through a communication medium 805 such as a modem, an interne connection, a telephone connection, or a wired or wireless commumcation signal carrier wave. In some cases, the computer may be connected to a server 809 or other communication device for relaying information from a user to the computer or from the computer to a user. In some cases, the user may store data or information obtained from the computer through a communication medium 805 on media, such as removable media 812. It is envisioned that data or diagnosese can be transmitted over such networks or connections for reception and/or review by a party. The receiving party can be, but is not limited to, an individual, a health care provider, or a health care manager. In one embodiment, a computer- readable medium includes a medium suitable for transmission of a result of an analysis of a biological sample, such as a level of one or more biomarker. The medium can include a result regarding a diagnosis of having a taste or smell disorder for a subject, wherein such a result is derived using the methods described herein.

[00392] Sample information can be entered into a database for the purpose of one or more of the following: inventory tracking, assay result tracking, order tracking, subject management, subject service, billing, and sales. Sample information may include, but is not limited to: subject name, unique subject identification, subject-associated medical professional, indicated assay or assays, assay results, adequacy status, indicated adequacy tests, medical history of the subject, preliminary diagnosis, suspected diagnosis, sample history, insurance provider, medical provider, third party testing center or any information suitable for storage in a database. Sample history may include but is not limited to: age of the sample, type of sample, method of acquisition, method of storage, or method of transport.

[00393] The database may be accessible by a subject, medical professional, insurance provider, third party, or any individual or entity granted access. Database access may take the form of electronic communication such as a computer or telephone. The database may be accessed through an intermediary such as a customer service representative, business representative, consultant, independent testing center, or medical professional. The availability or degree of database access or sample information, such as assay results, may change upon payment of a fee for products and sendees rendered or to be rendered. The degree of database access or sample information may be restricted to comply with generally accepted or legal requirements for patient or subject confidentiality.

Examples

[00394] While some embodiments have been shown and described herin, such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein can be employed in practicing the invention.

Example 1: IL-6 in Hyposmia

[00395] CONTEXT: IL-6 levels can be elevated in plasma of patients with both acute and chronic diseases but levels have not been measured in patients with smell loss (hyposmia). [00396] OBJECTIVE: To determine IL-6 levels in patients with smell loss (hyposmia).

[00397] STUDY DESIGN: This is a retrospective study of patients who presented for evaluation of hyposmia. All measurements were made without reference to the origin of any collected sample.

[00398] METHODS: IL-6 was measured in plasma, urine, saliva and nasal mucus in 59 patients with hyposmia of several etiologies and compared with levels measured in normal subjects. Measurements were made by use of a 96 plate spectrophotometric ELISA assay.

[00399] RESULTS: IL-6 was present in all biological fluids studied. Mean (SEM) levels in plasma, saliva, and nasal mucus in patients were significantly higher than in controls (0.95 [0.10] vs 0.12 [0.03] pg/mL, 0.57 [0.05] vs 0.30 [0.01] pg/mL, and 29.7 [3.8] vs 11.6 [0.5] pg/mL, respectively; all P<0.001). The concentration of IL-6 in nasal mucus in patients was significantly higher than in controls and was more than 30 times higher than in any other biological fluid. Mean (SEM) levels in urine were not significantly different: 0.92 (0.17) pg/mL for patients and 1.26 (0.41) pg/mL for controles (P>0.50). Compared with controls, IL-6 in patients was significantly elevated in plasma, saliva, and nasal mucus.

[00400] CONCLUSION AND RELEVANCE: Elevated IL-6 in patients with hyposmia compared to normal subjects is reported herein. Because IL-6 is a pro -inflammatory cytokine, these changes can relate to local or systemic inflammatory processes which play roles either as a cause of or as a result of the pathological processes associated with hyposmia. These studies support the concept that hyposmia has a biochemical basis and that discovery of the role(s) IL-6 plays in hyposmia offers an opportunity to learn more about the biochemical pathology underlying hyposmia and to establish new methods to treat the associated changes.

[00401] Introduction

[00402] Loss of smell (hyposmia) can be a symptom reflective of multiple chronic disease processes involving multiple organ systems including endocrine, vitamin, trace metal, metabolic, neurological, neurodegenerative, hematological, immunological and other organ systems.

Hyposmia can reflect both local changes in the oral or nasal cavities affecting olfactory receptors, in the nerves connecting receptors to the brain or in the brain itself. The systemic changes associated with the major pathologies noted above can include hyposmia as a major symptom.

[00403] This study attempts to understand the multiple pathologies responsible for initiation and perpetuation of hyposmia by studying changes in secretions of the multiple organ systems in which hyposmia occurs. Specific biochemical moieties are associated with hyposmia onset and that their replacement has corrected this symptom. For example, lack of thyroid hormone can induce hypothyroidism with its associated systemic symptoms, one of which can be hyposmia; administration of thyroid hormone can correct the systemic symptoms of hypothyroidism and the associated hyposmia. Zinc deficiency can induce multiple systemic symptoms and hyposmia which can be manifested by decreased gustin [carbonic anhydrase (CA) VI] secretion;

administration of zinc ion to zinc deficient patients can correct both these systemic symptoms and the associated hyposmia associated with increased CA VI secretion.

[00404] The multiple biochemical moieties of these diverse organ systems which correct hyposmia in these various pathologies can be growth factors which stimulate olfactory epithelial stem cells to initiate maturation and renewal of the sensory cells responsible for normal olfaction to occur.

[00405] Olfaction is a complex process comprised of multiple components including receptors, nerves and brain. The local and systemic components of this complex process have not been fully explored. Cell signaling processes can be critical in any complex sensory system such as olfaction and can involve adenylyl cyclases, sonic hedgehog and cytokines. Here, levels of IL-6 in patients with hyposmia are investigated since no prior studies of this type among these patients have been reported.

[00406] IL-6, a proinflammatory cytokine, can be over produced in a spectrum of clinical illnesses and conditions including cardiovascular disease, osteoporosis, arthritis, Type II diabetes, renal disease, hepatitis, schizophrenia, preeclampsia, various neoplasms, periodontal disease, frailty, stress and functional decline. In these conditions, increased IL-6 can be found in blood plasma. IL-6 can be increased in both plasma and ventricular fluid following acute but not chronic head injury. Increased IL-6 can be found in cerebrospinal fluid following traumatic brain injury and can trigger nerve growth factor secretion in astrocytes. Increased IL-6 can be found in blood plasma of patients with persistent sciatic pain and IL-6 mR A can be increased in rat spinal cord following peripheral nerve injury. Increased IL-6 can be found in plasma and in saliva of some patients with burning mouth syndrome (BMS); no IL-6 differences were reported in these patients with and without associated depression and perceived pain. However, stress hormones can regulate IL-6 expression in various ovarian carcinoma cells through a Src- dependent mechanism. Both specific and nonspecific factors can elicit changes in IL-6 in several biological fluids in several disease processes including neurological, inflammatory and psychological stress.

[00407] To evaluate IL-6 in olfaction, IL-6 levels were investigated in plasma, urine, parotid saliva and nasal mucus among patients with hyposmia and were compared to similar

measurements obtained in a group of normal subjects. [00408] Methods

[00409] SUBJECTS: Subjects of the study were 59 patients, 26 men, 33 women, age 10-86y, 54±2y (Mean±SEM) who presented with various degrees of smell loss. All studies were performed in a prospective manner. Diagnoses of these patients included 24 with post-influenza- like hyposmia and hypogeusia (PIHH), seven with allergic rhinitis, seven with congenital smell loss, six with hyposmia related to idiopathic causes, five with head injury, four with drug induced hyposmia, three with phantageusia and hyposmia and three with hyposmia and BMS. All patients had loss of smell as manifested by subjective statements and by olfactometry measurements in which impaired smell function was determined in each patient. Olfactometry was performed using psychophysical techniques with four odorants (pyridine, nitrobenzene, thiophene and amyl acetate). These techniques have been validated by performance in a double- blind clinical trial. Olfactory impairment was determined by impaired detection thresholds (DT) and/or recognition thresholds (RT) (elevated above normal) and/or decreased magnitude estimation (ME) levels (below normal levels) for one or more of the four odorants.

[00410] By use of these techniques, smell loss was confirmed in each patient with 12 patients exhibiting Type I hyposmia (the most severe form of hyposmia with RTs = 0 and MEs = 0 for all patients for all odors), 44 patients with Type II hyposmia (the next most severe form of hyposmia with DTs, RTs and MEs < normal for all patients) and 3 patients exhibiting Type III hyposmia (the least severe form of hyposmia with DTs and RTs = normal but MEs < normal).

[00411] Subjects of this study also included nine normal volunteers: 5 men, 4 women, age 39- 76y, 60±8y. All normal subjects were healthy and not taking any prescribed medications. All volunteers had normal smell function by subjective statements and by normal olfactometry.

[00412] PROCEDURES : At initial clinical evaluation, blood plasma was collected from each patient by venipuncture, placed in ice into tubes containing ΙΟΟμΙ of zinc free heparin, centrifuged at 3000 rpm for 10 min, the plasma removed and stored at -20°C until assayed.

Urine from each patient was collected over a 24-hour period in timed relationship to collection of blood plasma. Urine volume was measured and a 20ml aliquot was stored at 4°C until assayed. Parotid saliva was collected from each patient immediately after blood collection by placement of a Lashley cup over Stensen's duct with lingual stimulation with reconstituted lemon juice (Borden, Real Lemon, Stamford, CT). Saliva was collected in ice in plastic tubes over an 8-12 min period. Samples were stored at -20°C until assayed. Nasal mucus was collected from each patient directly from the nasal cavity in 50ml wide mouth plastic tubes over a period of two-five days in timed relationship to collection of blood, urine and saliva. After each daily collection nasal mucus was stored at 4°C. After total collection nasal mucus was transferred to plastic centrifuge tubes, centrifuged at 18K-20K x g for 40-55 min, the supernatant transferred to plastic PCR tubes and stored at -20°C until assayed.

[00413] Similar collections of blood, urine, saliva and nasal mucus were also obtained from each normal volunteer.

[00414] IL-6 was measured by a spectrophotometric 96 plate ELISA assay obtained from R&D Systems (Minneapolis, MN). Tests were employed following the manufacturer's directions. Since measurements of IL-6 in nasal mucus were not previously performed various sample dilutions were developed to perform the assay. These studies reflect all measurements of IL-6 in these biological fluids made among patients. IL-6 measurements were compared at various time periods of collection and storage without significant deterioration of IL-6 values.

[00415] All measurements were made without reference to origin of any collected sample. After all measurements were completed, values were matched with patients' records, sorted by diagnosis and compared to results obtained in normal subjects. Mean±SEM were determined for each patient diagnostic category and compared to similar results in normals. Differences were determined by Student t test with /?<0.05 considered significant. Analysis of variance among patient diagnosis, smell loss type (I, II, III) and IL-6 levels in each biological fluid studied was performed with the Tukey-Kramer technique with /?<0.05 considered significant.

RESULTS

[00416] Levels of IL-6 were measured in all biological fluids studied. Comparison of IL-6 levels in each biological fluid were compared between patients and normalsIL-6 levels in patients demonstrated large, consistent and significant differences (Table I). IL-6 in plasma, saliva and nasal mucus in patients was significantly higher than in normals. Mean nasal mucus levels in patients were 2.6 times that in normals, mean saliva levels were 1.9 times that in normals and mean plasma levels were 7.9 times that in normals.

[00417] Comparison of IL-6 among patients categorized by etiology of loss with normals demonstrated that mean plasma IL-6 was significantly higher in all patients compared to normal controls; the highest level was found in BMS, the next in head injury, the third highest in PIHH and the lowest in patients with allergic rhinitis (Table II). Mean urine IL-6 in patients was similar to normals in all patient categories except congenital hyposmia in whom levels were significantly lower than in normal controls. Mean saliva IL-6 was significantly above normal controls in patients with BMS, head injury and PIHH with the highest level in BMS. Mean nasal mucus IL-6 was elevated in patients with head injury, BMS, allergic rhinitis, phantageusia and PIHH but significantly only in patients with BMS and PIHH. IL-6 levels in nasal mucus were highest in patients with head injury and BMS. [00418] Comparative analysis of IL-6 levels in normal controls in plasma, urine, saliva and nasal mucus revealed a specific hierarchy (Table I) different from that found in patients. Levels of IL- 6 in nasal mucus were higher than in any other biological fluid being over 10 times that found in urine, saliva or in blood plasma. Levels were next highest in urine, then saliva and lowest in plasma. The ratio of nasal mucus:plasma was 97: 1 , of nasal mucus:saliva 34: 1 and nasal mucus :urine 9: 1.

[00419] Comparative analysis of IL-6 levels in patients also yielded a hierarchy of levels but with a somewhat different set of ratios than that found in normals. The highest level of IL-6 was also found in nasal mucus which was over 30 times the levels found in saliva, urine or in plasma. The next highest levels were found in plasma and urine (levels were similar) and the lowest level in saliva. The ratio of nasal mucus:plasma was 31 : 1 about 1/3 that found in normals, nasal mucus:saliva was 52: 1, about 1½ times the ratio in normals and nasal mucus:urine was 32: 1, about 3½ times the ratio found in normals.

[00420] TABLE I: IL-6 IN PLASMA, URINE, PAROTID SALIVA AND NASAL

MUCUS IN PATIENTS WITH HYPOSMIA AND IN NORMAL SUBJECTS

I nter leu kin 6 Level,

Mean (SEM), pg/mL

NORMALS PATIENTS

P Value

BIOLOGICAL FLUIDS IL-6 (n=9) IL-6 (n=59)

PLASMA 0.12 (0.03) 0.95 (0.10) 0.001

URINE 1.26 (0.41) 0.92 (0.17) >0.5

SALIVA 0.30 (0.01) 0.57 (0.05) 0.001

NASAL MUCUS 11.6 (0.5) 29.7 (3.8) 0.001

[00421] TABLE II: IL-6 IN PLASMA, URINE, PAROTID SALIVA AND NASAL MUCUS IN PATIENTS WITH HYPOSMIA

PIHH (n=24) 1.03 <0.001 1.22 >0.05 0.51 >0.05 29.7 <0.005 (0.15) (0.38) (0.05) (5.2)

Allergic 0.62 <0.001^a 0.71 >0.05 0.39 >0.05 39.5 >0.05 Rhinitis (0.12) (0.16) (0.06) (18.2)

(n=7)

Congenital 0.72 <0.001^a 0.29 <0.025^b 0.59 >0.05 19.6 >0.05^a (n=7) (0.15) (0.06) (0.14) (8.3)

Idiopathic 1.03 <0.001 1.00 >0.05 0.55 >0.05 13.6 >0.05^a (n=6) (0.15) (0.40) (0.07) (4.7)

Head Injury 1.47 <0.001 0.83 >0.05 0.68 <0.02 54.4 >0.05 (n=5) (0.56) (0.26) (0.18) (23.7)

Drug 0.70 <0.001^a'^b 0.78 >0.05 0.53 >0.05^c 10.8 >0.05^d

Induced (0.10) (0.26) (0.05) (4.0)

(n=4)

Phantogeusia 0.56 <0.001^a 0.44 >0.05 0.45 >0.05 31.5 >0.05^a (n=3) (0.08) (0.21) (0.13) (23.0)

BMS (n=3) 2.20 <0.001 0.90 >0.05 1.40 <0.001 50.9 <0.005

(0.6) (0.50) (0.70) (11.5)

Control 0.12 Ref. 1.26 Ref. 0.30 Ref. 11.6 Ref. (n=9) (0.03) (0.41) (0.01) (0.50)

Abbreviations: BMS, burning mouth syndrome; IL-6, interleukin 6; PIHH, postinflu

hyposmia and hypogeusia; Ref, reference.

a P<0.05 vs. BMS.

b. P<0.02 vs. PIHH.

c P<0.01 vs. PIHH.

d PO.005 vs. BMS.

[00422] There were no significant differences among IL-6 levels in plasma, saliva or nasal mucus with smell loss type and patient diagnosis (determined by ANOVA, results not shown). Discussion

[00423] The present study is the first to demonstrate IL-6 in patients with hyposmia and IL-6 elevations among patients with hyposmia compared to normal subjects. If these findings were to relate to similar results found in rheumatoid arthritis (RA) then elevated IL-6 could be considered a causal factor for initiation of hyposmia reflective of local and/or systemic immunological and/or inflammatory changes in blood, saliva or nasal mucus. This hypothesis is consistent with finding smell loss among patients with inflammatory RA. Among patients with hyposmia, chronic lymphocytic inflammation can be found in nasal mucous membranes of patients with PIHH. Elevated IL-6 can be found in nasal lavage fluid from patients with naturally acquired viral rhinitis. Paravirus and other viruses can be found in turbinate epithelial cells of patients with post viral olfactory dysfunction. Manifestation of herpes virus infection can be found in olfactory bulb neurons in mice as long as 200 days after they were initially experimentally infected as well as in astrocytes in the suspected portal of entry. However, histological changes which may occur in the olfactory epithelium, transmitting nerves or in the brain under these conditions have not been investigated. Treatment of RA with IL-6 inhibitors has been associated with diminution of both inflammation and IL-6 elevation. If IL-6 elevation in hyposmic patients were related to the cause of their pathology then treatment with IL-6 inhibitory drugs might be associated with improvement of their smell function.

[00424] Elevation of many substances locally or systemically can inhibit smell function including zinc, cadmium, drugs of several types, and several other chemical moieties. Elevated IL-6 could act as an endogenous substance regulating olfactory neuronal activity because it can regulate neuronal and glial cell activity. Thus, IL-6 elevations among patients with hyposmia and chronic head injury may relate to neurological as well as to inflammatory changes.

[00425] Finding elevated levels of IL-6 in both plasma and in saliva in some patients with BMS suggests not only a response to an inflammatory process but also a possible neurological process as well. Suggestion of a neurological rather than an inflammatory mechanism responsible for the pyrosis in BMS is consistent with the lack of obvious signs of oral inflammation among these patients. BMS can be considered a trigeminal small fibre neuropathy and treatment with an antioxidant, GABAergic drugs or repetitive transcranial magnetic stimulation can be used to alleviate this condition.

[00426] Results of the present study also illustrate that IL-6 levels in nasal mucus are higher than those in plasma, urine or saliva. This appears to be the first direct comparison of IL-6 levels in these biological fluids and the first demonstration that levels of IL-6 in nasal mucus in patients with hyposmia and in normal subjects are increased relative to that in plasma, urine or saliva. This finding is logically consistent with the abundance of microbial and antimicrobial agents normally present in nasal mucus. Active inflammatory agents in nasal mucus can include bacteria, viruses, fungi, and other substances including histamine whereas anti-inflammatory agents found include lysozyme, lactoferrin and albumin. However, contrary to this supposition, the highest level of IL-6 in nasal mucus reported here was not in patients with allergic rhinitis in which these agents might be expected to be most active but in patients with head injury and BMS in whom no active local nasal inflammatory process presumably occurs although both patient groups exhibit hyposmia. [00427] Possible mechanism(s) of the relationship(s) of IL-6 signaling to loss of smell are multiple. IL-6 can be part of a complex and sophisticated signaling system which plays multiple roles in body metabolism. IL-6 can be an inflammatory cytokine which drives acute phase proteins including C-reactive protein and fibrinogen, both proteins induced by systemic inflammation. It can be a factor in differentiation of B cells into antibody producing plasma cells. It can influence NF-κΒ and ATP - ubiquitin-dependent proteolytic pathways, it can activate TNF-a and thereby activate apoptotic pathways which could directly inhibit smell function. Neuropoietin, an IL-6 related cytokine which affects signaling through ciliary neurotrophic factor receptor, could directly inhibit smell function since inhibition of several ciliary factors have induced smell loss in patients with Kartagener's and Bardet-Biedl syndromes. Patients with Castleman's disease can overproduce IL-6 and treatment which inhibits either IL-6 or IL-6 receptor activity can alleviate symptoms of the disease. 11-6 deficient mice are incapable of mounting an inflammatory response. After binding to its receptor, the IL-6 receptor complex activates gp 130 signaling in cells that would not normally express IL-6 receptor, a mechanism that can play a role in pathophysiology of chronic inflammatory disorders.

[00428] These results suggest that both specific and nonspecific factors may increase IL-6 in both acute and chronic disease processes. These processes can include neurological and inflammatory processes and processes involving psychological stress. Indeed, stress hormones can regulate IL-6 expression in various ovarian carcinoma cells through a Src-dependent mechanism.

[00429] This is the first study of any type in which IL-6 measurements were obtained and compared in patients with chronic disease processes in several biological fluids (plasma, urine, saliva and nasal mucus) in a similar timed based study. These results offer an insight into the signaling processes present among some patients with hyposmia which may influence some of the complex processes responsible for their sensory changes.

Example 2: Cytokine changes in nasal mucus and other biological fluids in patients with smell loss classified by age.

[00430] Cytokine changes in nasal mucus and other biological fluids in patients with smell loss classified by age

[00431] BACKGROUND : Cytokine activity in nasal mucus has not been studied in patients with smell loss (hyposmia). Cytokines have been reported to change with age with some known to increase, others to decrease and others not to change. However, most of these reported changes were observed in relationship to stimulated activities mainly in various hematological system functions. Therefore, we performed a survey of cytokine activity in several biological fluids including nasal mucus in patients with hyposmia classified by age.

[00432] METHODS: By use of sensitive 96 plate spectrophotometric ELISA techniques IL-l , IL-Ιβ, IL-lra, IL-1 RII, IL-2, IL-2R, IL-6, IL-10, IL-18, TNF-a, IFN-β and IFN-γ were measured in nasal mucus, blood plasma, urine and parotid saliva in 79 subjects with hyposmia at progressive 10 year age groups from <30y to >70y.

[00433] RESULTS: IL-lra levels in nasal mucus were the highest found in any biological fluid consistent with the role of this cytokine as an anti- inflammatory factor. Cytokines IL-l , IL-Ιβ and IFN-γ were present in nasal mucus consistent with their roles as inflammatory factors.

These latter cytokines were absent in blood plasma, urine and saliva.

[00434] CONCLUSIONS : Cytokine levels in nasal mucus suggest a complex interaction occurs between proinflammatory and anti-inflammatory cytokines among patients with hyposmia with the highest levels in the anti- inflammatory cytokine IL-lra in nasal mucus. Cytokine levels varied with age in complex patterns. This is the first demonstration of several cytokines in nasal mucus in relationship to other biological fluids.

[00435] INTRODUCTION

[00436] Cytokines are cell signaling moieties activated by specific stimuli which lead to many physiological responses. However, these signaling proteins function in such multiple pathways that their specificity may not be clearly defined. We have been interested in the roles various cytokines play in patients with smell loss (hyposmia) and have published preliminary data related to changes that occur in cytokines with age among these patients who also exhibit anorexia and taste distortions.

[00437] However, the pathology associated with hyposmia is quite varied in relationship to multiple clinical conditions. Most patients develop hyposmia following a viral-type infection whereas others develop this symptom following head injury or associated with systemic and nasal symptoms of allergic rhinitis. We have attempted to determine some common biochemical threads underlying these diverse pathologies and in so doing we and others before us determined that smell loss has been attributed to changes in secretions from multiple organ systems including decreased levels of trace metals and vitamins, treatment with various therapeutic drugs and associated with various pathological conditions, including diabetes, other endocrine disorders, neurological disorders and liver disease. In an effort to define these putative common pathological threads underlying these various pathologies we undertook systematic studies of the multiple biochemical parameters putatively responsible for loss of smell function. To perform these studies we evaluated levels of trace metals in blood plasma and cyclic nucleotides in saliva and nasal mucus. These studies revealed that many patients with hyposmia exhibited lower than normal levels of zinc in their saliva and lower than normal levels of adenylyl cyclases in their saliva and nasal mucus.

[00438] Because hyposmia involves changes in sensory receptors, nerves and brain it was apparent that changes in cell signaling and thus, in cytokines, were involved in this complex system. For example, inhibition of sonic hedgehog secretion initiated loss of taste by inhibiting stem cell stimulation in taste buds which is responsible for growth and maturation of the elegant repertoire of cellular components initiating and maintaining normal taste function. Because cytokines play such a significant role in cell signaling we undertook a survey of several cytokines in blood plasma, urine, saliva and nasal mucus among a group of patients with hyposmia.

[00439] Cytokine levels have been previously reported by many investigators to change with age. In- vitro production of IL-Ιβ, IL-6, TNF-a and IFN-γ by peripheral mononuclear cells was reported increased in aged compared to younger human subjects. Stimulated T cells from aged mice compared to young mice showed increased production of IFN-γ, decreased IL-2 but no differences in IL-4. Leukocytes from elderly humans produced higher amounts of IL-1, IL-6, IL-8 and TNF-a than from younger subjects, there was a decreased release of IL-2 and soluble IL-2R but IL-2R expression in the cell surface was not increased in the elderly. No age related differences were observed in absolute amounts of IL-1 β and IL-6 after normalizing for circulatory monocytes and there was no age related decline in IL-2. Because of these results changes in cytokine levels by age appeared to be one factor by which changes in hyposmia might occur.

[00440] Results of our studies indicated that in patients with hyposmia levels of the antiinflammatory cytokine IL-lra in nasal mucus were higher than in any other cytokine contrasted with the presence of lesser but still large amounts of nasal mucus proinflammatory cytokines. These results suggest that a complex interplay between anti- and proinflammatory cytokines occurs among these patients and may play a role in their smell function.

[00441] MATERIALS AND METHODS

[00442] Patients: Subjects were 79 patients who presented to The Taste and Smell Clinic, Washington, DC with clinical complaints of smell loss. Other than smell loss patients were well and healthy. Patients were 44 women and 35 men, aged 53±5y (Mean±SEM), range 21-83y. Studies were approved by the Institutional Review Board of the Georgetown University Medical Center; all patients gave informed consent to participate in this study. [00443] At initial clinical evaluation blood plasma was obtained by venipuncture and stored at - 20°C until assayed. A 24-hour urine was collected in direct timed relationship to collection of blood plasma; volume was measured and an aliquot stored at -20°C until assayed. Parotid saliva was collected from each patient immediately after blood collection by placement of a modified Lashley cup over Stensen's duct with lingual stimulation with reconstituted lemon juice (Borden, Real Lemon, Stamford, CT) and stored at -20°C until assayed. Nasal mucus was collected using spontaneous nasal discharge over two-five days, as previously described. After each daily collection samples were stored at 4°C. Samples were transferred to plastic tubes, centrifuged at 17K-19K x g for 40-55 min and the supernatant stored at -20°C until assayed.

[00444] Cytokines and some of their receptors (IL-l , IL-Ιβ, IL-lra, IL-1 RII, IL-2, IL-2R, IL-6, IL-10, IL-18, TNF-a, IFN-β and IFN-γ) were measured by sensitive spectrophotometric 96 plate ELISA assays obtained from R&D Systems (Minneapolis, MN). Tests were employed following the manufacturer's directions. Since no prior measurements of cytokines in nasal mucus were performed various sample dilutions had to be developed to perform this assay.

[00445] All measurements were made without reference to any specific clinical data including patient age. After all measurements were completed values were matched with patient records and sorted by age. Mean±SEM were determined for each cytokine with patients separated into progressive age groups of <30y, 31-40y, 41-50y, 51-60y, 61-70y, and >70y. Significance of differences were determined by Student t test with /?<0.05 considered significant.

[00446] RESULTS

[00447] IL-l . Values were obtained only in nasal mucus and saliva (Table 3). Levels of nasal mucus ranged from 24-92 times levels in saliva. For nasal mucus there was a small, gradual decrease with age. There was no apparent age related relationship observed with age in saliva.

[00448] IL-Ιβ. Values were obtained only in nasal mucus (Table 3). Values in nasal mucus were generally higher than those found for IL-la. There were no apparent age related relationships observed with age.

[00449] IL-lra. Values were obtained in each biological fluid (Table 3). Levels in nasal mucus were the highest found in any biological fluid. Levels in nasal mucus ranged from 80 to over 1000 times higher than those in plasma which were comparatively the lowest among fluids studied. IL-lra levels in nasal mucus were almost 1000 times higher than levels in IL-la and IL-Ιβ. By age there was an inverted U shaped pattern for nasal mucus with the peak at age 40- 49y. There was a U shaped pattern in saliva with the nadir at a similar age with nasal mucus, 40- 49y. [00450] IL-1 RII. Values were obtained in each biological fluid (Table 3). Levels in nasal mucus were about 0.1-0.5% levels in plasma but 5-90 times levels in saliva and 2-9 times the level in urine. Levels in nasal mucus were about 0.2-0.4% values measured in IL-lra but 5-17 times levels measured in IL-la and varied from 5-17 times higher than levels of IL-Ιβ. By age there was an inverted U shaped pattern for nasal mucus similar to that measured in IL-lra with the peak at age 40-49y and a similar inverted U shaped pattern in plasma with a peak at a similar age.

[00451] IL-2. At dilutions used for this cytokine values were not obtained in any fluid.

[00452] IL-2R. Values were measured in all biological fluids (Table 1). Levels in nasal mucus varied from 2%-36% below that measured in plasma, from 2%-20% below that measured in urine but from 5-335 times higher than that measured in saliva. Levels in nasal mucus were lower than those measured in IL-l , IL-Ιβ, IL-lra or IL-1 RII. There was a relative inverted U shaped pattern with age in nasal mucus with the peak again at age 40-49y.

[00453] IL-6. Values were measured in all biological fluids (Table 4). Levels in nasal mucus were higher than in any other of these biological fluids being 3-13 times levels in plasma, from 7-25 times levels in urine and 3-10 times levels in saliva. Levels in IL-6 were lower than those in IL-la, IL-Ιβ, IL-lra, IL-1 RII and IL-2R. With respect to age levels in nasal mucus increased up to age 30-39y and decreased thereafter.

[00454] IL-10. Values were measured in nasal mucus, saliva and plasma but not in urine.

Levels in nasal mucus were generally higher than in either saliva or plasma except at either end of the age range. Levels in nasal mucus ranged from 2-20 times higher than in plasma and 2-8 times higher in saliva. With respect to age there was a gradual increase in nasal mucus until age 60-69y and then a decrease thereafter.

[00455] IL-18. Values were measured in nasal mucus, saliva and plasma but not in urine, as with IL-10. Values in nasal mucus varied with respect to levels in plasma but were generally higher than levels in saliva by as much as a factor of seven. Levels in nasal mucus were higher than in IL-2R, IL-6 or IL-10 but lower than in IL-la, IL-Ιβ, IL-lra or IL-1 RII. With respect to age there was again an inverted U shaped pattern in nasal mucus with a peak at age 50-59y.

[00456] TNF-q. Values were measured in all biological fluids (Table 4). Levels in nasal mucus were higher than in any of these biological fluids being 3-11 times higher than levels in plasma, 7-23 times levels in urine and 7-27 times levels in saliva. Levels of TNF-a in nasal mucus were higher than levels of IL-6 or IL-10 but lower than in IL-la, IL-Ιβ, IL-lra, IL-1 RII and IL-18. With respect to age there was an approximate U shaped pattern in nasal mucus with a nadir at age 40-49y albeit the lowest level was at age 60-69y; highest levels were measured at both ends of the age range.

[00457] IFN-β. Values were obtained in all biological fluids except for urine. Values in nasal mucus were generally similar to levels in plasma but higher than levels in saliva. Levels in nasal mucus were higher than levels in IL-2R, IL-6, IL-10, IL-18 or TNF-a but lower than levels in IL- la, IL-Ιβ, IL-lra and IL-1 RII. Age related values in nasal mucus and saliva cannot be evaluated due to multiple missing data but there appears to be an inverted U shaped pattern in plasma with the peak at age 30-39y.

[00458] IFN-γ. Values were obtained only in nasal mucus. Levels were higher than in IL-2R, IL-6, IL-10 and TNF-a but lower than levels in IL-la, IL-Ιβ, IL-lra and IL-1 RII. There may be a U shaped pattern with age with the nadir at age 50-59y.

[00459] TABLE 3: CHANGES IN HUMAN CYTOKINE LEVELS WITH AGE

30- 50 .6 0 0 65 0 0 0 1830 3908 148 307

39 5 3

40- 24 .7 0 0 38 0 0 0 3919 2445 6 506

49

50- 33 1.6 0 0 75 0 0 0 1214 6739 93 188

59 5 7

60- 32 1.7 0 0 57 0 0 0 1421 7722 35 959

69 8

>70 38 1.3 0 0 54 0 0 0 1374 9726 52 249

2 8

IL-1 RII (pg/mL) IL-2 (pg/mL) IL-2R (pg/mL)

Nas Nas Nasa

Age al Sali Plas Uri al Sali Plas Uri 1 Sali Plas Uri Muc va ma ne Muc va ma e Muc va ma e us us us y

<30 674* 123 1232 394 0 0 0 0 9 0 467 101

3 9

30- 1844 59 1533 302 0 0 0 0 27 16 808 988

39 8

40- 3003 43 3668 352 0 0 0 0 335 1 939 169

49 0 8

50- 1961 36 1649 478 0 0 0 0 31 16 814 116

59 5 7

60- 2153 24 2146 366 0 0 0 0 80 19 1418 117

69 7 7

>70 1087 40 2025 302 0 0 0 0 27 12 1002 120

7 3

<30 245† 4 68 121 0 0 0 0 9 0 107 679

30- 849 21 1699 52 0 0 0 0 20 11 89 255

39 40- 1518 20 1114 64 0 0 0 0 188 1 523 454

49 3

50- 1075 13 2311 117 0 0 0 0 17 10 153 275

59

60- 1023 7 2921 66 0 0 0 0 48 18 312 216

69

>70 288 18 2684 77 0 0 0 0 15 10 132 71

* Mean

† ±SEM

0 Values were 0

[00460] TABLE 4: CHANGES IN HUMAN CYTOKINE LEVELS WITH AGE

IL-6 (pg/mL) IL-10 (pg/mL) IL-18 (pg/mL)

Nas Nas Nasa

<30 1.1 * 0.1 0.08 0.0 0.03 0.5 0.10 0 60 18 175 0

1 5 2

30- 2.7 0.0 0.10 0.0 0.32 0.4 0.53 0 167 36 187 0

39 8 6 8^a

40- 1.7 0.1 0.19 0.2 0.84 0.4 0.40 0 122 92 195 0

49 5 5 4^a

50- 0.3 0.0 0.07 — 2.47 0.3 0.62 0 405 54 224 0

59 7 3

60- 0.7 0.0 0.22 0.0 8.99 1.0 0.48 0 116 117 163 0

69 6 4 8

>70 0.3 0.0 0.09 — 0.42 0.5 0.45 0 119 59 338 0

6 3

<30 0.6† 0.0 0.01 0.0 0.10 0.1 0.01 0 46 10 116 0

2 1 4 30- 0.14 0.0 0.02 0.0 0.22 0.1 0.10 0 74 10 62 0

39 2 1 0

40- 1.0 0.0 0.09 0.1 0.56 0.0 0.11 0 80 27 54 0

49 9 2 3

50- 0.1 0.0 0.04 — 0.40 0.0 0.12 0 193 26 90 0

59 4 1

60- 0.12 0.0 0.08 0.0 6.80 0.2 0.03 0 41 37 32 0

69 3 2 4

>70 0.08 0.0 0.03 — 0.02 0.2 0.06 0 53 32 136 0

3 2

TNF-a (pg/mL) IFN-β (pg/mL) IFN-γ (pg/mL)

Nas Nas Nasa

<30 11.7 0.8 1.0 0.7 0 0 1584 0 109 0 0 0

30- 8.9 0.3 3.0 0.5 0 0 2175 0 77 0 0 0

39

40- 5.8 0.6 3.0 0.3 1239 8 1690 0 67 0 0 0

49

50- 7.3 0.6 2.2 0.4 — 160 — 0 55 0 0 0

59

60- 2.7 0.4 2.0 0.4 351 6 492 0 121 0 0 0

69

>70 16.1 0.6 3.2 0.7 428 15 533 0 63 0 0 0

<30 5.3† 0.8 0.1 0.3 0 0 996 0 12 0 0 0

30- 5.2 0.1 0.7 0.1 0 0 2175 0 8 0 0 0

39 40- 2.2 0.3 0.9 0.1 819 8 1 142 0 31 0 0 0

49

50- 2.7 0.1 0.8 0.1 — 101 — 0 21 0 0 0

59

60- 2.7 0.1 0.9 0.3 309 4 381 0 69 0 0 0

69

>70 6.7 0.3 0.5 0.2 428 15 483 0 63 0 0 0

* Mean

† ±SEM

0 Values were 0

— No sample obtained

a p<0.05 with respect to age 60-69y

[00461] All numbers in Table 3 and Table 4 that are within a single cell should be read as one number.

[00462] DISCUSSION

[00463] These data indicate that multiple cytokines in multiple biological fluids are present in patients with hyposmia but levels of each cytokine vary in each fluid. Levels in nasal mucus were generally the highest measured in any biological fluid with a specific pattern of activity. This pattern appears related to a specific interplay between proinflammatory cytokines (e.g., IL- l , IL-Ι β, IL-6, IL-18, TNF-a) and anti- inflammatory cytokines (e.g., IL-lra, IL-10, IFN-γ) among hyposmic patients and suggest that while there are multiple etiological factors responsible for loss of smell, many of which have no apparent inflammatory component, e.g., following head injury or hypothyroidism, there may be an underlying physiological interplay among these nasal mucus cytokines.

[00464] Cytokine changes in nasal mucus suggest this complex interplay between

proinflammatory cytokines and their competitive inhibitor anti-inflammatory components among patients with hyposmia. Since changes in nasal mucus can and do reflect changes in olfactory function these results are relevant to basic mechanisms underlying smell loss in these patients. The identities of proinflammatory cytokines in nasal mucus are consistent with the anatomical and pathological changes of chronic inflammation in the nasal mucus membranes as previously identified among these hyposmic patients. However, these results are contrasted with levels of IL-lra, the competitive inhibitor of these proinflammatory cytokines, which are much higher in concentration than those of the proinflammatory cytokines suggesting an endogenous protective effect against acute or chronic inflammation among these patients. This result is consistent with treatment in hyposmic patients with theophylline or other phosphodiesterase inhibitors which improve smell function among these patients and also inhibiting secretion of TNF-a and other proinflammatory cytokines.

[00465] Cytokines are pleiotropic and redundant molecules with a wide variety of functions with overlapping activities in several cells. For example, TNF-a was initially considered to have mainly immunomodulatory and proinflammatory effects but more recent data suggest that TNF-a also has significant anti- inflammatory properties. On the other hand IL-10 inhibits synthesis of proinflammatory cytokines including IL-1, IL-6 and TNF-a by modulating lipo saccharide induced fever and similar changes in animals.

[00466] IL-1 is a 17 KD proinflammatory cytokine synthesized from a variety of cell types associated with disease states or during perturbations such as immune responses. It is part of a family of cytokines which share a conserved β-trefoil structure which binds to receptors belonging to the IL-1 receptor family. In most instances in which inflammation is activated IL-1 is the major protagonist. IL-18, usually considered a proinflammatory cytokine, has also been reported to play an antagonistic role to this activity of IL-1 but this action is still controversial. There is a naturally occurring IL-1 specific receptor antagonist, IL-lra, which shares 40% amino acid homology with IL-Ιβ, binds to IL-1 surface receptors with the same affinity as IL-1, does not possess agonist activity but acts as a competitive inhibitor of IL-1. Studies suggest that IL-1 plays a key role in triggering the cascade of inflammatory cellular responses with IL-lra blocking endogenous IL activity.

[00467] IL-lra in nasal mucus is the highest secreted cytokine among all biological fluid cytokines measured and highest among all the nasal mucus cytokines measured. This level in nasal mucus is about 800 times higher than the levels of IL-1 a, over 400 times higher than the level of IL-Ιβ, about 30 times the level of IL-1 RII and over 19000 times higher than the level of TNF-a.

[00468] With this context in mind evaluation of results of this study suggests a complex interplay occurs in nasal mucus in patients with hyposmia between proinflammatory cytokines IL-1 a, IL-Ιβ, IL-6, IL-18 and TNF-a and their competitive inhibitors IL-lra, IL-10 and IFN-γ. These results suggest a mucosal balance between proinflammatory and anti-inflammatory cytokines among these patients suggesting some control of these elements in hyposmia. This contrasts with the imbalance between these proinflammatory and anti- inflammatory cytokines in patients with inflammatory bowel disease in which IL-1 is significantly greater than IL-lra which has been related to a novel mechanism of chronic intestinal inflammation in chronic inflammatory bowel disease and the presence of hyposmia. [00469] The increased proinflammatory cytokines in nasal mucus is consistent with the anatomical and pathological changes of inflammation in the nasal mucus membranes previously identified among patients with viral rhinitis and among these hyposmic patients. Theophylline treatment, which improved smell function among these patients, also inhibited secretion of TNF- α and other proinflammatory cytokines which support these observations. Since levels of IL- lra, the major competitive inhibitor of these proinflammatory cytokines are much higher than those of the proinflammatory cytokines these results suggest an endogenous protective effect against acute or chronic inflammation occurs among these patients.

[00470] While the changes observed in this study relate to mucosal changes in cytokines released into nasal mucus by cells in nasal epithelial glands these changes may also relate in changes in central nervous system and anterior pituitary function. Changes in IL-Ιβ, IL-lra and IL-10 gene expression have all been shown to be increased during systemic inflammation in the central nervous system and anterior pituitary. These results suggest that IL-lra may be secreted by the anterior pituitary as a systemic anti- inflammatory hormone released in response to IL-Ιβ from multiple system sources consistent with the results we have observed among hyposmic patients. IL-18 gene polymorphism has also been found among patients with allergic diseases.

[00471] Changes we measured in cytokine levels with age in each biological fluid are complex. Previous investigators reported that many factors influence cytokine changes in addition to age including caloric restriction, endotoxin presence, oxidative stress and hormonal states. Age changes in TNF-a have been reported to be predictive of insulin resistance. Thus, differences in levels in each biological fluid we studied may reflect not only differences in each cytokine with age but also other mechanisms related to multiple factors not identified in this study. Indeed, the most relevant aspect of this study relates not to age changes but to changes in cytokines in nasal mucus itself relative to changes in other biological fluids as related to patients with hyposmia. With these cytokine changes in nasal mucus these results suggest a complex interplay between proinflammatory cytokines and their competitive inhibitors occurs among patients with hyposmia. Since changes in nasal mucus can and do reflect changes in olfactory function these results are relevant to the basic mechanism(s) underlying smell loss in this group of patients. Indeed, antibodies to specific cytokines have been successful in disease treatment.

[00472] Among the cytokines measured there is an apparent hierarchy of levels among the various biological fluids studied. Nasal mucus IL-lra is the most prevalent cytokine in any biological fluid studied although there are significant variations in these measurements. Urinary IL-lra is the most prevalent urinary cytokine among all urinary cytokines studied. Plasma and saliva IL-1 RII are the most prevalent cytokines among the plasma and saliva levels measured. [00473] Cytokine concentrations found in these biological fluids are relative to measurement techniques used. Because these results reflect cytokine levels under physiological conditions, albeit in patients with hyposmia, it is difficult to compare these results with those previously reported by most other investigators since they mainly reported age related changes in cytokine activity in hematological or tissue cell function in response to specific stimulatory and inhibitory substances. Inamizu, et al. reported macrophage IL-Ιβ production decreased with age whereas in our studies there was no change with age in nasal mucus. Previous investigators reported decreased keratinocyte IL-l production with age whereas our results show a generalized increase with age in IL-l in each biological fluid studied. It is known that IL-1 stimulates IL- lra production but the complex changes with age we demonstrate in either nasal mucus or saliva do not support the observation that IL-1 relates to increased levels in IL-lra. IL-2R from older subjects have been reported to decline with age but in our studies there were increases in plasma, little change in urine and a complex pattern of change in nasal mucus. Beharka, et al. reported that IL-6 production does not increase with age whereas in our studies IL-6 in plasma increased, particularly at age 60-69y, and in urine at age 49-49y. IL-10 in our study increased with age in each fluid whereas Ye, et al. reported age-related declines in IL-10 in brain sections and glial cells in mice. Consistent with our studies, aged marine CD4⁺ T cells produced more IL-10 than did young cells. There were no changes reported in IFN-γ production with age whereas we reported an increase in nasal mucus levels at age 60-69y. Variable age related changes in TNF-a and interferon have been reported by many previous investigators. Changes in cytokine levels with age in several other biological fluids have been previously reported; Kawasaki, et al. reported that RANKL and OPG levels decreased in gingival crevicular fluid with age and Yamakawa, et al. reported an increase in IL-Ιβ production in murine parotid acinar cells with age and they reported a decrease in IL-6 .

[00474] This is the first study in which cytokine levels in several biological fluids in patients with hyposmia have been reported and the first in which cytokine levels in several biological fluids, measured in near time to each other, have been reported.

Example 3: IgE and Eosinophil Changes in Hyposmia

[00475] Patients with smell loss have multiple clinical and biochemical characteristics which define their pathology. In a recent reanalysis of data obtained at The Taste and Smell Clinic in Washington, DC 28 patients were analyzed with taste and smell dysfunction. Each of the 28 patients has a significant abnormality in ability to taste and smell. Eight of the 28 patients (29%) have an elevated serum IgE level. Levels range from 128-781 kU/L (mean±SEM, 258±82). Four of these patients have a primary diagnosis of post influenza- like hyposmia and hypogeusia (PIHH); four have a primary diagnosis of allergic rhinitis. Patients are seven men, one woman, aged 35-71y. Five of the 28 patients (18%) have an elevated plasma eosinophil level. Levels range from 3.7 - 11.9% of total white blood cells (mean±SEM, 6.1±2.3%) with eosinophil counts of 307-750 cells (mean±SEM, 462±176). Three of these patients have a diagnosis of PIHH, two have a diagnosis of allergic rhinitis. Patients are four men, one woman, aged 39-71y. Two have both an elevated IgE and eosinophil count, one man, one woman, both with PIHH, age 39 and 71, respectively. These patients also have low levels of cAMP and cGMP in their saliva and nasal mucus.

[00476] Patients are treated and tested with theophylline to test if restoration of smell function can be restored. A comparison of this subpopulaton of patients to larger group of patients is performed.

Example 4: Role of Nitric Oxide in Smell Loss

[00477] In this example the role of nitric oxide in smell loss is investigated. Phosphodiesterase (PDE) inhibitors may improve smell loss through other mechanisms, such as through nitric oxide (NO). Theophylline, a generalized PDE inhibitor, may increase nitric oxide (NO) at the same time that it increases cAMP and cGMP. Patients with hyposmia treated with theophylline may not only have increased nasal mucus cAMP and cGMP but also increased NO. NO through guanylate cyclase produces cGMP, whose elevation mediates, in part, NO stimulatory effects on smell loss. cGMP may be 1) degraded by PDE iso forms and 2) may be enhanced by PDE inhibition which maintains its presence.

[00478] To investigate this possibility, treatments with alternative pharmaceutical compositions, which may increase nitric oxide production, levels, effective amounts or half life are

administered to patients with hyposmia. Pharmaceutical compositions that are directed towards increase in cGMP and cAMP may also be administered.

Example 5: Intranasal Administration of theophylline

[00479] Objective: To determine whether intranasal theophylline methylpropyl paraben can correct hyposmia and hypogeusia.

[00480] Design: We performed an open-label pilot study in patients with hyposmia and hypogeusia under the following 3 conditions: (1) before treatment, (2) after oral theophylline treatment, and (3) after intranasal theophylline treatment. Under each condition, we performed subjective evaluations of taste and smell functions, quantitative measurements of taste (gustometry) and smell (olfactometry), and measurements of serum theophylline level and body weight.

[00481] Patients: Ten patients with hyposmia and hypogeusia clinically related to the effects of viral illness, allergic rhinitis, traumatic brain injury, congenital hyposmia, and other chronic disease processes were selected.

[00482] Interventions: Oral theophylline methylpropyl paraben, 200 to 800 mg/d for 2 to 12 months, was administered to each patient. This treatment was discontinued for 3 weeks to 4 months when intranasal theophylline methylpropyl paraben, 20 pg/d in each naris, was administered for 4 weeks.

[00483] Main Outcome Measures: At termination of each condition, taste and smell function was determined subjectively, by means of gustometry and olfactometry, with measurement of serum theophylline levels and body weight.

[00484] Results: Oral theophylline treatment improved taste and smell acuity in 6 patients after 2 to 12 months of treatment. Intranasal theophylline treatment improved taste and smell acuity in 8 patients after 4 weeks, with improvement greater than after oral administration. No adverse effects accompanied intranasal drug use. Body weight increased with each treatment but was greater after intranasal than after oral administration.

[00485] Conclusions: Intranasal theophylline treatment is safer and more effective in improving hyposmia and hypogeusia than oral theophylline treatment.

[00486] Loss of smell (hyposmia) and taste (hypogeusia) are common symptoms that affect many thousands of patients in the United States, as reported by several investigators. Effective treatment for these symptoms has been demonstrated only recently and has not been formally established.

[00487] Before effective treatment to correct loss of smell and taste can be established, a biochemical basis for the cause of these symptoms is necessary. To accomplish this, we determined that these symptoms are commonly caused by decreased secretion of several growth factors in the saliva and nasal mucus. The growth factors act on stem cells in taste buds and olfactory epithelial cells to generate the elegant repertoire of cellular components in these sensory organs. Growth factor stimulation of these sensory organs is thought to maintain normal taste and smell function. If these growth factors were diminished by any of several diseases and pathological conditions, then hyposmia and hypogeusia occur. These conditions and diseases include trace metal deficiencies; vitamin deficiencies; liver disease; diabetes mellitus's; other metabolic, otolaryngo logical, and neurodegenerative disorders, including multiple sclerosis, Parkinson disease, and Alzheimer disease; and other neurological disorders. Effective treatment to increase secretion of these growth factors is therefore necessary to improve hypogeusia and hyposmia and return taste and smell function to normal as demonstrated by several previous studies.

[00488] To understand more about these processes, a comprehensive study of many patients with loss of smell and taste determined that levels of the salivary and nasal mucus growth factors cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) were lower than in healthy subjects and were responsible for the onset of hyposmia and hypogeusia in many of these patients. Indeed, as hyposmia increased in severity, levels of these salivary and nasal mucus growth factors decreased in a consistent manner.

[00489] To increase salivary and nasal mucus cAMP and cGMP levels and thereby correct hypogeusia and hyposmia, we hypothesized that treatment with a phosphodiesterase inhibitor would be useful. To test this hypothesis, a previous study from our institution administered oral theophylline to 312 patients with hyposmia and hypogeusia in an open- label controlled clinical trial. Results of this study demonstrated that oral theophylline treatment successfully corrected hyposmia in more than 50% of these patients. Subsequent investigators have used other oral phosphodiesterase inhibitors to correct hyposmia. An open-label study also demonstrated that, as nasal mucus cAMP and cGMP levels increased, hyposmia was corrected, whereas in patients in whom these moieties did not increase, hyposmia was not corrected. These results suggested that some patients may be resistant to treatment with oral theophylline.

[00490] However, successful treatment with oral theophylline that increased nasal mucus levels of cAMP and cGMP required increased theophylline doses, sometimes prolonged treatment duration, and endurance of adverse effects, including restlessness, gastrointestinal tract discomfort, sleep difficulties, tachycardia, and other unwanted symptoms. Theophylline treatment also required regular determinations of blood theophylline levels to ensure adequate drug absorption and lack of toxic effects. These efforts limited use of this orally administered drug.

[00491] Because of these adverse effects, we wished to learn more about the pharmacology of theophylline administration. After treatment with oral theophylline, the drug was found in blood, nasal mucus, and saliva in a dose dependent manner. These results were consistent with improvement in smell function as demonstrated in patients with hyposmia in the prior clinical trial. Results of these studies and efforts to improve therapeutic efficacy and reduce adverse effects of oral theophylline administration made it logical to administer the drug intranasally. In this manner, the drug could affect olfactory receptors more directly without causing the systemic adverse effects associated with oral therapy. [00492] To accomplish this, with assistance of an established medical device company, an intranasal delivery device was developed. With assistance of an established pharmaceutical company, the drug was packaged for sterile, intranasal delivery. Using this device, an open-label, single source, controlled pilot study in 10 patients with hyposmia and hypogeusia and with levels of parotid saliva and nasal mucus cAMP and cGMP below the reference range was performed to determine safety and to compare smell and taste responses after intranasal theophylline treatment, with patient responses before any treatment and after oral theophylline treatment.

[00493] METHODS

[00494] Patients

[00495] We selected 10 patients with hyposmia and hypogeusia from the 312 patients who participated in the prior open-label controlled clinical trial for this pilot study. Each patient had undergone previous evaluation before any drug treatment, followed by treatment with oral theophylline. These patients had hyposmia and hypogeusia and exhibited levels of cAMP and cGMP lower than their respective reference ranges in the saliva and nasal mucus before theophylline treatment. These 10 patients were selected from the group undergoing previous evaluation and treatment for the intranasal trial because (1) their response to oral theophylline was subjectively submaximal; (2) they developed adverse effects after attempts to increase the drug dose to obtain a more maximal clinical response, thus limiting the administered drug dose; and (3) they resided in an area in close proximity to The Clinic, which made their frequent return visits to The Clinic more practical for any additional clinical trial.

[00496] These 10 patients included 7 men, aged 37 to 77 (mean [SEM] age, 64 [6]) years, and 3 women, aged 47 to 77 (62 [1 1]) years. Patients had 1 of the following 5 different clinical causes of sensory dysfunction: allergic rhinitis (n=3), postinfluenzalike hyposmia and hypogeusia (n =3), head injury (n=2), congenital hyposmia49 (n =1), and other disorders (n=l). Patients served as their own control throughout each condition of this study. The conditions included no treatment (before entry into the oral theophylline study), oral theophylline treatment, and intranasal theophylline treatment.

[00497] Procedures

[00498] Subjective changes in smell and taste function under each study condition were measured by questionnaire before measurements of smell or taste function. Responses were graded on a scale from 0 to 100, with 0 reflecting no subjective response in overall sensory function; 100, return to normal sensory function; and values between 0 and 100 intermediate responses. Overall sensory function was defined as the ability to smell all odors and identify all tastants, although response intensity varied. [00499] Smell and taste functions under each study condition were measured by standardized psychophysical sensory testing techniques. Measurements included determination of detection thresholds (DTs), recognition thresholds (RTs), magnitude estimation (ME), and hedonic response (HR) for 4 odors (i.e., pyridine [dead fish] , nitrobenzene [bitter almond], thiophene [petroleum], and amyl acetate [banana oil]) (olfactometry) and for 4 tastants (i.e., sodium chloride [salt] , sucrose [sweet] , hydrochloride [sour], and urea [bitter]) (gustometry). These techniques have been previously described with olfactometry confirmed in a prior controlled double-blind clinical trial. Each measurement was performed independent of any prior knowledge of response.

[00500] Serum theophylline levels were measured by fluorescence polarization at each treatment condition. Body weight was measured with a calibrated clinical scale during each study condition and reported at the final measurement in each study condition.

[00501] Study Protocol

[00502] The patients each underwent initial clinical evaluation at The Clinic to establish the cause, degree, and character of hyposmia and hypogeusia exhibited. Measurements in blood, urine, erythrocytes, saliva, and nasal mucus determined before their entry into the open trial of oral theophylline established the biochemical cause of their hyposmia and hypogeusia to be related to their levels of saliva and nasal mucus cAMP and cGMP being lower than the reference range. These 10 patients were then selected for this study on the basis of the laboratory and clinical criteria noted previously.

[00503] The 10 patients in this intranasal pilot study entered into the previous oral theophylline study according to a protocol approved by the institutional review board of the Georgetown University Medical Center. In this prior trial, oral theophylline was administered daily in 2 divided doses (at breakfast and lunch) of 200, 400, 600, or 800 mg for 2 to 12 months of treatment. Treatment was divided into 2- to 4-month periods, at which time patients returned to The Clinic for measurements of subjective sensory responses, olfactometry, gustometry, serum theophylline level, and body weight. If oral theophylline treatment failed to correct hyposmia at a given dose, the theophylline dose was increased by 200 mg, and the patient underwent reevaluation at 2- to 4-month intervals to a dose of 800 mg. As noted previously, study patients did not obtain a maximal clinical response to oral theophylline' or, while taking oral theophylline at a given dose, demonstrated some clinical improvement but experienced significant adverse effects that limited increasing the oral dose as necessary to achieve maximum clinical benefit. In the 10 patients selected for the intranasal pilot study, oral theophylline treatment was discontinued 3 weeks to 4 months before initiation of the intranasal drug trial. At that time, the mean (SEM) serum theophylline level was unmeasurable in any patient (0 [0] mg/L).

[00504] A pilot study of intranasal theophylline treatment was then initiated among these 10 patients. This trial was an investigator initiated phase 1, open- label, single-source, controlled pilot study, intranasal drug therapy reflected a compassionate trial of a potentially more useful therapeutic method to improve hyposmia (and hypogeusia) than oral theophylline. Before the intranasal trial, risks and benefits were explained and the patients signed an informed consent.

[00505] The intranasal administration device was a calibrated 1 mL syringe fitted with a nozzle that fit comfortably into the anterior naris (Wolfe Tory Medical, Inc) and loaded under sterile conditions with 20 pg of theophylline methylpropyl paraben in a 0.4-mL saline solution

(Foundation Care). Patients were instructed to direct the spray superiorly into the nasal cavity but not posteriorly into the nasopharynx. This technique was practiced before study initiation with sterile saline. Each patient used the technique easily and as demonstrated before drug

administration.

[00506] Each patient delivered the theophylline dose in each naris once daily throughout the study. Patients underwent evaluation 1, 2, and 4 weeks during drug use with the same

measurements used for the oral study.

[00507] Values for the oral trial were taken from the last measurements made before

discontinuation of oral drug treatment and before initiation of the intranasal trial. This period varied from 2 to 12 months after oral treatment initiation and reflected the maximal improvement in sensory function each patient experienced. Values for the intranasal pilot study were taken from measurements obtained after completion of 4 weeks of intranasal treatment.

[00508] The mean and standard error of the mean for all values obtained at each study condition were compared. Differences were considered significant if P < .05 by the unpaired t test. Paired comparison tests were also used with differences considered significant if P < .05 by the t test.

[00509] RESULTS

[00510] With oral theophylline administration, hypogeusia improved after 2 to 12 months of treatment, but hypogeusia improved further within 1 to 4 weeks of intranasal treatment (Figure 4). Results of gustometry after oral and intranasal theophylline are shown in Figure 4. Before treatment, DTs for sucrose, hydrochloride, and urea (less sensitive) and RTs for all tastants were elevated (less sensitive) above the reference levels. Magnitude estimations for all tastants were lower (less sensitive) than the reference level. Hedonic responses for sodium chloride, hydrochloride, and urea were lower (less unpleasant) than the reference levels. After oral theophylline treatment, DTs for sucrose and hydrochloride and RTs for sodium chloride, hydrochloride, and urea decreased (more sensitive). Magnitude estimations for all tastants increased (more sensitive) and HR for hydrochloride and urea increased (more unpleasant) as previously reported. After intranasal theophylline treatment, DTs and RTs for all tastants were lower (more sensitive) than before treatment or after oral theophylline treatment. Magnitude estimations for all tastants after intranasal theophylline treatment were higher (more intense) than before any treatment or after oral theophylline treatment. Hedonic responses for sodium chloride, hydrochloride, and urea were more negative (more unpleasant), whereas HRs for sucrose were more positive (more pleasant) than before any treatment or after oral theophylline treatment.

[00511] After oral theophylline treatment, hyposmia improved with 2 to 12 months of treatment but improved more with intranasal theophylline after 1 to 4 weeks of treatment (Figure 5).

Olfactometry comparisons of oral and intranasal theophylline treatment are shown in Figure 5. Before treatment, compared with reference levels, DTs and RTs for all odorants were elevated (less sensitive); MEs for all odorants were decreased (less sensitive); HRs for pyridine and thiophene were decreased (less unpleasant); and HRs for nitrobenzene and amyl acetate were decreased (less pleasant). After oral theophylline treatment, DTs and RTs for all odorants were decreased (more sensitive), MEs for all odorants were increased (more sensitive), and HRs for all odorants increased (for pyridine and thiophene, more unpleasant; for nitrobenzene and amyl acetate, more pleasant) as previously reported. After intranasal theophylline treatment, DTs and RTs for each odor were lower (more sensitive) than before treatment or after oral theophylline treatment. Magnitude estimations for each odor were higher (more intense) than before treatment or after oral theophylline treatment. Hedonic responses to thiophene were more negative (more unpleasant) and to nitrobenzene were more positive (more pleasant) than before treatment or after oral theophylline treatment.

[00512] Smell and taste acuity were reported to be subjectively improved with oral theophylline treatment, but greater improvement was reported after 4 weeks of intranasal theophylline treatment. After oral theophylline treatment, 6 patients reported overall increased taste and smell function, whereas 4 reported no improvement. After intranasal theophylline treatment, 8 of the 10 patients reported overall improvement in taste and smell functions, whereas 2 reported no improvement. This response frequency is higher than that previously reported among patients with hyposmia and treated with oral theophylline, in which slightly more than 50% reported improvement.

[00513] Taste and smell acuity were measured as subjectively improved after oral theophylline treatment, but this improvement was measured as increased after 4 weeks of intranasal theophylline treatment (Figure 6). After intranasal theophylline treatment, a 2-fold improvement was measured for taste and smell functions compared with oral treatment. Paired t test results showed that responses after intranasal theophylline were significantly greater than after oral theophylline treatment (taste, P < .05; smell, P < .025).

[00514] Body weight increased from pretreatment levels after oral theophylline treatment, but weight increased more after intranasal theophylline treatment. After oral theophylline treatment, mean (SEM) weight increased by 1.5 (0.4) kg from pretreatment values, whereas after intranasal theophylline treatment, weight increased by 2.5 (0.5) kg from pretreatment values. Patients related this change to increased food flavor obtained by improved smell function after intranasal theophylline treatment, which increased appetite and food enjoyment, resulting in subsequent weight gain. These changes were measured in each patient group despite no sensory

improvement in 4 patients after oral theophylline treatment and none in 2 after intranasal theophylline treatment.

[00515] During oral theophylline treatment, the mean (SEM) serum theophylline level at the time of maximum improvement for these 10 patients was 6.4 (2.0) mg/L (to convert to micromoles per liter, multiply by 5.55). During intranasal theophylline treatment, the mean serum theophylline level was 0.0 (0.0). Discontinuation of intranasal theophylline treatment resulted in loss of smell and taste function within 1 week in 2 patients and after 6 weeks in 2. Four patients reported some persistence of improvement after 10 weeks.

[00516] COMMENT

[00517] Results of this open-label, single-source, controlled pilot trial demonstrate that oral theophylline effectively improved hyposmia, as previously reported. The earliest this improvement was measured was after 2 months of treatment, but maximal improvement varied from 4 to 12 months. These results also demonstrate that oral theophylline was effective in improving hypogeusia in the same time frame as improvement in smell acuity.

[00518] In addition, intranasal theophylline was shown to be safe and more effective than oral theophylline in correcting hyposmia and hypogeusia. This improvement was measured as early as 1 week after starting treatment, but maximal improvement varied from 1 to 4 weeks.

[00519] Mechanisms by which intranasal theophylline was more effective than oral theophylline are not clearly defined. Intranasal drug delivery avoids the first-pass hepatic effect of an oral drug, bypassing initial cytochrome P450 metabolism and decreasing metabolism of the orally administered drug, thereby allowing for lower intranasally administered drug doses to be clinically efficacious. This lowering of the drug dose from a range of 200 to 800 mg orally to 40 pg intranasally was sufficient and specific enough to also avoid production of systemic adverse effects. This delivery mechanism may also avoid development of drug resistance that has occurred with oral theophylline. In addition, because more drug presumably contacts the olfactory epithelium with intranasal than with oral theophylline, direct nasal administration may activate more olfactory receptors than does oral administration.

[00520] However, additional actions of intranasal theophylline might enhance its therapeutic efficacy. Theophylline has been shown to inhibit symptoms of allergic rhinitis, which affected 3 patients in the intranasal trial. Many of the diseases and conditions that caused hyposmia and hypogeusia have an associated inflammatory component that may be suppressed by the antiinflammatory effects of a phosphodiesterase inhibitor. In addition, drugs introduced intranasally can be delivered into the brain (1) directly by absorption through the cribriform plate along the olfactory bulb, (2) indirectly by absorption through blood-brain barrier receptors, or (3) through combinations of both methods. Although studies of theophylline absorption from nasal mucus into the brain have not been performed, studies of insulin, nerve growth factor, several neurotransmitters, and other moieties indicate uptake of these intranasally introduced moieties into the brain.

[00521] Whatever its mechanism of action, intranasal theophylline in this pilot study corrected hyposmia and hypogeusia relatively rapidly in 8 of 10 patients with several clinical diagnoses. The 2 patients who did not experience improvement were men, one with allergic rhinitis and the other with the effects of viral illness.

[00522] These results are consistent with prior studies in which several intranasal drugs were more effective than oral drugs. Inhaled adrenocorticosteroids were more effective with fewer adverse effects for asthma treatment than oral adrenocorticosteroids, and inhaled

adrenocorticosteroids were more efficacious in asthma treatment than oral prednisolone acetate. Intranasal zolmitriptan achieved faster control of migraine headaches with fewer effects than the orally administered drug. Nasal administration of chicken type II collagen suppressed adjuvant arthritis in rats more effectively than oral administration.

[00523] However, intranasally administered drugs have also been reported to be only as effective as these same drugs given orally. Intranasal estradiol valerate was as effective as oral administration in alleviating postmenopausal symptoms but produced less frequent mastalgia and uterine bleeding. Intranasal desmopressin acetate was as effective for nocturnal enuresis as the oral drug but at a dose one-tenth that of the oral drug. Intranasal desmopressin is the preferred route for management of central diabetes insipidus. Example 6. Nasal mucus transplantation

[00524] In this example, a subject with a smell disorder (e.g., hyposmia or anosmia) is treated by nasal mucus transplantation. A donor is selected who has been screened for normal olfactory function by standardized psychophysical sensory testing techniques. Measurements include determination of detection thresholds (DTs), recognition thresholds (RTs), magnitude estimation (ME), and hedonic response (HR) for 4 odors (i.e., pyridine [dead fish] , nitrobenzene [bitter almond], thiophene [petroleum], and amyl acetate [banana oil]) (olfactometry). A nasal mucus sample of about 4 mL in volume is harvested from the screened donor and prepared in a clinical environment for transplantation to the recipient's nasal cavity. Treatment efficacy is evaluated by administering the standardized psychophysical sensory testing techniques before and after transplantation. The transplantation technique is repeated as necessary according to the efficacy testing data. Recipient's olfactory ability improves according to the standardized psychophysical sensory testing.

Example 7. Saliva transplantation

[00525] In this example, a subject with a taste disorder (e.g., hypogeusia or ageusia) is treated by saliva transplantation. A donor is selected who has been screened for normal taste function by standardized psychophysical sensory testing techniques. Measurements include determination of detection thresholds (DTs), recognition thresholds (RTs), magnitude estimation (ME), and hedonic response (HR) for 4 tastants (i.e., sodium chloride [salt] , sucrose [sweet] ,

hydrochloride [sour], and urea [bitter]) (gustometry). A saliva sample of about 4 mL in volume is harvested from the screened donor and prepared in a clinical environment for transplantation to the recipient's oral cavity. Treatment efficacy is evaluated by administering the standardized psychophysical sensory testing techniques before and after transplantation. The transplantation technique is repeated as necessary according to the efficacy testing data. Recipient's ability to taste improves according to the standardized psychophysical sensory testing.

Example 8. Treatment with antibody inhibitors of IL-6

[00526] In this example, a subject with a taste or smell disorder is treated by administration of an effective amount of an inhibitory antibody against IL-6 or a receptor of IL-6. The inhibitory antibody can be tociluzumab, sarilumab, elsilimomab, siltuximab, sirukumab, BMS-945429,

CDP6038, VX30, ARGX-109, or FM101. The administration is by intranasal administration.

Treatment efficacy is evaluated by administering the standardized psychophysical sensory testing techniques before and after administration of the antibody. The administration is repeated as necessary according to the efficacy testing data. Recipient's ability to taste and smell improves according to the standardized psychophysical sensory testing. [00527] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

-I l l-

Claims

WHAT IS CLAIMED IS:

1. A method of diagnosing a taste or smell disorder in a subject, the method comprising:

(a) obtaining a biological sample from the subject;

(b) measuring a level of one or more biomarkers present in the biological sample, wherein the one or more biomarkers are selected from the group consisting of IL-l , IL- 1β, IL-lra, IL-1 RII, IL-2, IL-2R, IL-6, IL-10, IL-18, IFN-β, IFN-γ, IgE, eosinophils, and any combination thereof; and

(c) diagnosing the subject with the taste or smell disorder when the level(s) of the one or more biomarkers are abnormal.

2. The method of claim 1, further comprising treating the subject diagnosed with the taste or smell disorder.

3. The method of claim 1 or 2, wherein the diagnosing in (c) is computer

implemented.

4. The method of any one of claims 1-3, further comprising sending a result from the diagnosing to a party via a communication medium.

5. The method of any one of claims 1-4, wherein the diagnosing comprises determining that the level of the one or more biomarkers, individually, is one selected from the group consisting of: above a threshold level, below a threshold level, and within a range that is indicative of having taste or smell disorders.

6. The method of claim 5, wherein the threshold level is an average level as measured in a control population.

7. The method of claim 5, wherein the threshold level is at least 1.5 times higher or lower than an average level as measured in a control population.

8. The method of any one of claims 1-7, wherein the biological sample comprises a blood sample, a plasma sample, a urine sample, a saliva sample, a nasal mucus sample, or a combination thereof.

9. The method of any one of claims 1-8, wherein the measuring comprises measuring an eosinophil level, and the diagnosing comprises determining that the eosinophil level is above a threshold level.

10. The method of claim 9, wherein the biological sample comprises a blood sample or a plasma sample, and wherein the threshold level is from 300 cells/HPF (high powered field) to 400 cells/HPF.

11. The method of claim 9, wherein the biological sample comprises a blood sample or a plasma sample, and wherein the threshold level is about: 300 cells/HPF, 350 cells/HPF, or 400 cells/HPF.

12. The method of any one of claim 9-11, wherein measuring the eosiniphil level is performed with a Coulter counter.

13. The method of any one of claim 1-12, wherein the measuring comprises measuring an IgE level, and the diagnosing comprises determining that the IgE level is above a threshold level.

14. The method of claim 13, wherein the biological sample comprises a blood sample or a plasma sample, and wherein the threshold level is from 75 kU/L to 125 kU/L.

15. The method of claim 13, wherein the biological sample comprises a blood sample or a plasma sample, and wherein the threshold level is about: 75 kU/L, 100 kU/L, or 125 kU/L.

16. The method of any one of claim 13-15, wherein measuring the IgE level comprises a fluorescence polarization assay.

17. The method of any one of claim 1-16, wherein the measuring comprises measuring a level of one or more biomarkers selected from the group consisting of IL-la, IL-Ιβ, IL-6, IL-18, and any combination thereof, and the diagnosing comprises determining that level(s) are above a threshold level.

18. The method of claim 17, comprising measuring the IL-6 level.

19. The method of claim 18, wherein the biological sample is a nasal mucus sample, and wherein the threshold level is from 5 pg/mL to about 15 pg/mL.

20. The method of claim 18, wherein the biological sample is a plasma sample, and wherein the threshold level is from 0.05 pg/mL to about 0.2 pg/mL.

21. The method of claim 18, wherein the biological sample is a saliva sample, and wherein the threshold level is from 0.15 pg/mL to about 0.4 pg/mL.

22. The method of any one of claim 1-21, wherein the measuring comprises measuring a level of one or more biomarkers selected from the group consisting of IL-lra, IL-10, IFN-γ, and any combination thereof, and the diagnosing comprises determining that level(s) are, individually, below a threshold level.

23. The method of any one of claims 1-4, comprising diagnosing the subject with the taste or smell disorder based upon one or more measurements comprising:

(i) the level of IL-la that is about: 125 pg/mL to 195 pg/mL, 150 pg/mL to 170 pg/mL, 120 pg/mL to 170 pg/mL, or 150 pg/mL to 195 pg/mL; (ii) the level of IL-Ιβ that is about: 195 pg/mL to 300 pg/mL, 220 pg/mL to 275 pg/mL, 220 pg/mL to 300 pg/mL, or 195 pg/mL to 275 pg/mL;

(iii) the level of IL-lra that is about: 30,000 pg/mL to 90,000 pg/mL, 45,000 pg/mL to 75,000 pg/mL, 45,000 pg/mL to 90,000 pg/mL, or 30,000 pg/mL to 75,000 pg/mL;

(iv) the level of IL-1 RII that is about: 960 pg/mL to 2600 pg/mL, 1370 pg/mL to 2190 pg/mL, 1370 pg/mL to 2600 pg/mL, or 960 pg/mL to 2190 pg/mL;

(v) the level of IL-2 that is about: 0 pg/mL, 0.1 pg/mL, 0.2 pg/mL, 0.3 pg/mL, or 0.5 pg/mL;

(vi) the level of IL-2R that is about: 0 to 200 pg/mL, 50 to 150 pg/mL, 0 to 150 pg/mL, or 50 to 200 pg/mL;

(vii) the level of IL-6 that is about: 0.1 pg/mL to 2.2 pg/mL, 0.6 pg/mL to 1.7 pg/mL, 0.6 pg/mL to 2.2 pg/mL, or 0.1 pg/mL to 1.7 pg/mL;

(viii) the level of IL-10 that is about: 0 pg/mL to 3.5 pg/mL, 0.8 pg/mL to 2.7 pg/mL, 0.8 pg/mL to 3.5 pg/mL, or 0 pg/mL to 2.7 pg/mL;

(ix) the level of IL-18 that is about: 40 pg/mL to 290 pg/mL, 100 pg/mL to 230 pg/mL, 40 pg/mL to 230 pg/mL, or 100 pg/mL to 290 pg/mL;

(x) the level of IFN-β that is about: 0 pg/mL to 910 pg/mL, 230 pg/mL to 680 pg/mL, 230 pg/mL to 910 pg/mL, or 0 pg/mL to 680 pg/mL; or

(xi) the level of IFN-γ that is about: 55 pg/mL to 110 pg/mL, 70 pg/mL to 95 pg/mL, 70 pg/mL to 110 pg/mL, 55 pg/mL to 95 pg/mL.

24. The method of claim 23, wherein diagnosing is based upon 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 of the one or more measurements.

25. The method of any one of claims 1-24, wherein measuring comprises using one or more techniques that are fluorescence microscopy, a radioimmunoassay, a fluorescence immunoassay, fluorescence-activated cell sorting, mass spectrometry, liquid chromatography, electrophoresis, protein arrays, or a combination thereof.

26. The method of any one of claims 1-24, wherein measuring comprises using one or more antibodies that bind one or more biomarkers.

27. The method of claim 25, wherein at least one antibody is conjugated to an enzyme, a fluorescent molecule, or a radio-label.

28. The method of any one of claims 25-27, wherein the one or more antibodies are used in an immunostain, an immunoprecipitation, an Immunoelectrophoresis, an immunoblot, a western blot, a proximity ligation assay, or a spectrophotometry assay.

29. The method of claim 28, wherein the one or more antibodies are used in the spectrophotometry assay that is an EMIT (Enzyme Multiplied Immunoassay Technique) assay, an ELISA (Enzyme Linked Immunosorbent Assay), a sandwich ELISA, or a competitive ELISA.

30. The method of any one of claims 1-29, wherein the taste or smell disorder is anosmia, hyposmia, phantosmia, parosmia, ageusia, hypogeusia, phantageusia, or parageusia.

31. The method of claim 30, wherein the taste or smell disorder is anosmia or hyposmia.

32. The method of any one of claims 2-31 , wherein treating comprises administering a pharmaceutical composition comprising an effective amount of one or more phosphodiesterase inhibitors to the subject.

33. The method of claim 32, wherein the pharmaceutical composition is administered orally.

34. The method of claim 32, wherein the pharmaceutical composition is administered intranasally.

35. The method of any one of claims 32-34, wherein the one or more PDE inhibitors comprise a non-selective PDE inhibitor, a PDE-1 selective inhibitor, a PDE-2 selective inhibitor, a PDE-3 selective inhibitor, a PDE-4 selective inhibitor, a PDE-5 selective inhibitor, or a combination thereof.

36. A method of treating taste or smell disorders in a subject in need thereof, the method comprising administering an effective amount of an inhibitor of a pro -inflammatory cytokine.

37. The method of claim 36, wherein the inhibitor is an antibody, an antibody fragment, or an antibody mimetic.

38. The method of claim 37, wherein the antibody, antibody fragment, or antibody mimetic binds to the pro-inflammatory cytokine.

39. The method of claim 37, wherein the antibody, antibody fragment, or antibody mimetic binds to a receptor for the pro -inflammatory cytokine.

40. The method of claim 36, wherein the pro -inflammatory cytokine is IL-6 and the inhibitor is tociluzumab, sarilumab, elsilimomab, siltuximab, sirukumab, BMS-945429,

CDP6038, VX30, ARGX-109, FM101, or lunasin.

41. The method of claim 36, wherein the pro -inflammatory cytokine is IL-l and the inhibitor is IL-1RA.

42. The method of claim 36, wherein the pro -inflammatory cytokine IL-Ιβ and the inhibitor is canakinumab.

43. The method of claim 36, wherein the pro -inflammatory cytokine is TNF-a and the inhibitor is infliximab; adalimumab; certolizumab pegol; golimumab; etanercept; a xanthine derivative that is pentoxifylline; bupropion; or a 5-HT_2A agonist that is (R)-DOI (2,5-dimethoxy- 4-iodoamphetamine), TCB-2 (l-[(7R)-3-bromo-2,5-dimethoxybicyclo[4.2.0]octa-l,3,5-trien-7- yljmethanamine), LSD (lysergic acid diethylamide), or LSZ (Lysergic acid 2,4- dimethy lazetidide) .

44. A method of treating a taste or smell disorder in a subject in need thereof, the method comprising administering a pharmaceutical composition comprising an effective amount of an adenylyl cyclase activator, a guanylyl cyclase activator, a cAMP analog, a cGMP analog, or a combination thereof.

45. The method of claim 44, wherein the pharmaceutical composition comprises the adenylyl cyclase activator that is forskolin; 1,9-Dideoxyforskolin; 6-[3- (dimethylamino)propionyl]forskolin; adenylyl cyclase toxin; NB001 ; NKH 477; Pituitary adenylate cyclase activating polypeptide-38; Pituitary adenylate cyclase activating polypeptide- 27; or a combination thereof.

46. The method of any one of claims 44-45, wherein the pharmaceutical composition comprises the guanylyl cyclase activator that is A-50619 hydrochloride; atriopeptin II; 6β- Hydroxy-8,13-epoxy-labd-14-en-l 1-one; 9a-Hydroxy-8,13-epoxy-labd-14-en-l 1-one;

isoliquiritigenin; protoporphyrin IX; YC-1; BAY41-2272; CMF-1571; A-350619; BAY 41- 8543; BAY 63-2521; BAY58-2667; HMR1766; S3448; or a combination thereof.

47. A method of treating a taste or smell disorder in a subject in need thereof, the method comprising administering a pharmaceutical composition comprising an effective amount of one or more anti-inflammatory cytokines to the subject.

48. The method of claim 47, wherein the one or more anti- inflammatory cytokines comprise IL-lra, IL-10, IFN-γ, or a combination thereof.

49. The method of any one of claims 36-48, wherein the pharmaceutical composition is administered one, two, three, or more times per day.

50. The method of any one of claims 36-49, wherein the pharmaceutical composition is administered each day for from 7 days to 5 years, 7 days to 1 year, 7 days to 6 months, 7 days to 3 months, or 7 days to 1 month.

51. The method of any one of claims 36-49, wherein the pharmaceutical composition is administered each day on a continuous basis.

52. The method of any one of claims 36-51 , wherein the subject in need thereof experiences a decrease in a detection threshold (DT) score or a recognition threshold (RT) score for at least one tastant or odorant following administration of the pharmaceutical composition.

53. The method of any one of claims 36-52, wherein the subject experiences an increase in a magnitude estimation (ME) score for at least one tastant or odorant following administration of the pharmaceutical composition.

54. The method of any one of claims 36-53, wherein the subject in need thereof experiences a decrease in phantosmia, parosmia, phantageusia, or parageusia following administration of the pharmaceutical composition.

55. The method of any one of claims 36-54, wherein a pro -inflammatory cytokine level in the subject is lower following administration of the pharmaceutical composition.

56. The method of any one of claims 36-55, wherein eosinophil levels in the subject are lower following administration of the pharmaceutical composition.

57. The method of any one of claims 36-56, wherein IgE levels in the subject are lower following administration of the pharmaceutical composition.

58. The method of any one of claims 36-57, wherein a cyclic nucleotide level in the subject is higher following administration of the pharmaceutical composition.

59. The method of any one of claims 36-58, wherein the pharmaceutical composition is administered orally.

60. The method of any one of claims 36-58, wherein the pharmaceutical composition is administered intranasally.

61. The method of any one of claims 36-60, wherein the subject in need thereof was diagnosed with a taste or smell disorder according to the method of any one of claims 1-35.