US 2017007 1522A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2017/0071522 A1 PARSEY et al. (43) Pub. Date: Mar. 16, 2017

(54) METHOD OF DAGNOSING DEPRESSION A6B 5/55 (2006.01) BY PET IMAGING A6IB5/00 (2006.01) A6IB 6/03 (2006.01) (71) Applicants: Ramin PARSEY, East Setauket, NY A6IB 6/00 (2006.01) (US); Christine DELORENZO, Long (52) U.S. Cl. Island City, NY (US); Gregory CPC ...... A61 B 5/165 (2013.01); A61B 6/037 SULLIVAN, New York, NY (US) (2013.01); A61B 6/481 (2013.01); A61 B 5/055 (2013.01); A61 B 5/0042 (2013.01); A61B (72) Inventors: Ramin PARSEY, East Setauket, NY 6/501 (2013.01); A61B 6/5217 (2013.01); (US); Christine DELORENZO, Long Island City, NY (US); Gregory A61 K5I/0459 (2013.01) SULLIVAN, New York, NY (US) (57) ABSTRACT (73) Assignees: THE RESEARCH FOUNDATION FOR THE STATE UNIVERSITY OF NEW YORK, Albany, NY (US): THE The present invention provides a method of determining TRUSTEES OF COLUMBIA whether a subject is afflicted with a depressive disorder UNIVERSITY IN THE CITY OF comprising: NEW YORK, New York, NY (US) (i) introducing into the Subject a positron emission tomog raphy (PET) radioligand capable of binding with a (21) Appl. No.: 15/121,957 5-HT, receptor, (22) PCT Fed: Mar. 5, 2015 (ii) performing one or more PET scans of the subject; (iii) determining, by analysis of the one or more PET (86) PCT No.: PCT/US2O15/O18992 images, a receptor binding potential of the PET radio S 371 (c)(1), ligand for the serotonin 5-HT, receptor in a region of (2) Date: Aug. 26, 2016 interest in the subject; (iv) comparing the receptor binding potential value of the Related U.S. Application Data PET radioligand in the region of interest in the subject (60) Provisional application No. 61/949,743, filed on Mar. to a predetermined receptor binding potential threshold 7, 2014. value; and (V) classifying the Subject as having the depressive dis Publication Classification order or as not having the depressive disorder based on (51) Int. C. the comparison of step (iv), thereby determining A6 IB 5/16 (2006.01) whether the subject is afflicted with the depressive A6 IK 5L/04 (2006.01) disorder. Patent Application Publication Mar. 16, 2017. Sheet 1 of 2 US 2017/0071522 A1

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METHOD OF DAGNOSING DEPRESSION Tomography (PET) allow visualization and quantification of BY PET IMAGING serotonin receptor binding in vivo. 0007 PET involves detection of pairs of gamma rays 0001. This application claims priority of U.S. Provisional emitted indirectly by a positron-emitting radionuclide Application No. 61/949,743, filed Mar. 7, 2014, the contents (tracer) injected into the body. Images of tracer concentra of which are hereby incorporated by reference. tion in the body are then reconstructed by computer analysis. 0002 Throughout this application, certain publications Positron emitting isotopes include carbon, iodine, fluorine, are referenced in parentheses. Full citations for these pub nitrogen, and oxygen. These isotopes can replace their lications may be found immediately preceding the claims. non-radioactive counterparts in target compounds to pro The disclosures of these publications in their entireties are duce tracers that function biologically and are chemically hereby incorporated by reference into this application in identical to the original molecules for PET imaging, or can order to describe more fully the state of the art to which this be attached to said counterparts to give close analogues of invention relates. the respective parent molecule. Among these isotopes, F is 0003. The invention was made with government support a convenient labeling isotope due to its relatively long under Grant numbers MH40695, MH62185, 1MH074813, 109-minute half-life. and MHO90276 awarded by the National Institutes of Health. The government has certain rights in the invention. SUMMARY OF THE INVENTION BACKGROUND OF THE INVENTION 0008. The present invention provides a method of deter mining whether a subject is afflicted with a depressive 0004 Major depressive disorder (MDD) is a highly disorder comprising: prevalent psychiatric diagnosis that is associated with a high 0009 (i) introducing into the subject a positron emis degree of morbidity and mortality (Marangell et al., 2006; sion tomography (PET) radioligand capable of binding Merikangas et al., 2007; Woods, 2000). This debilitating with a serotonin 5-HT, receptor; disorder is currently one of the leading causes of disability 0.010 (ii) performing one or more PET scans of the nationwide among both medical and psychiatric conditions Subject; and is predicted to be the leading cause of disease burden by 0.011 (iii) determining, by analysis of the one or more the year 2030 (Lopez & Murray, 1998: World Health, 2004). PET images, a receptor binding potential of the PET 0005. There are currently 945 ways to meet diagnostic radioligand for the serotonin 5-HT" A receptor in a criteria for a major depressive episode and thus, patients region of interest in the subject; sharing as few as one common symptom can be diagnosed 0012 (iv) comparing the receptor binding potential with major depression. Further, diagnosis of MDD depends value of the PET radioligand in the region of interest in on the reliability of current diagnostic classifications and the Subject to a predetermined receptor binding poten (Subjective) structured diagnostic interviews (Karlsson et tial threshold value; and al., 2010). It is partly for this reason that, for several decades, 0013 (V) classifying the subject as having the depres epidemiological studies reported that women were twice as sive disorder or as not having the depressive disorder likely as men to develop MDD, with prevalence rates of 8% based on the comparison of step (iv), thereby deter and 4%, respectively (Jovanovic et al., 2008: Parker & mining whether the subject is afflicted with the depres Brotchie, 2010). Since men and women experience depres sive disorder. sion differently, these subjective criteria may have led to an 0014. The present invention also provides a method of under diagnosis of MDD in males. Consistent with this view, preparing a report classifying a subject as having a depres a 2013 study reported that when changes in case definitions sive disorder or as not having a depressive disorder which of MDD were implemented in a way that account for higher comprises: rates of anger, aggression and Substance abuse in men, MDD 0.015 (i) receiving the data of one or more PET scans prevalence estimates between sexes are eliminated (Martin of the subject performed by a PET imaging device after et al., 2013). a PET radioligand for a serotonin 5-HT, receptor was 0006. A biomarker is a characteristic that can be objec introduced into the subject; tively measured and used as an indicator of either normal or 0016 (ii) processing the data to determine a receptor pathogenic processes (Singh & Rose, 2009). Identification binding potential of the PET radioligand for the sero of psychiatric biomarkers for MDD would eliminate the tonin 5-HT, receptor in a region of interest in the need for Subjective diagnosis, and therefore help improve Subject and comparing the receptor binding potential diagnostic classification. Further, Such a marker may aid in better classifying the great heterogeneity observed across value to a predetermined receptor binding potential MDD presentation into more specific Sub-diagnostic catego threshold value; and ries as well as provide much needed evidence of the physi 0017 (iii) populating a report classifying the subject. ological underpinnings of MDD (Singh & Rose, 2009). Due 0018. The present invention further provides a method of to its role in MDD, the system could give rise treating a subject afflicted with a depressive disorder, com to a biomarker of depression. Previous research has impli prising cated the serotonergic system in MDD pathophysiology 0.019 (a) determining whether the subject is afflicted (Boldrini et al., 2008: Drevets et al., 1999; Parsey et al., with the depressive disorder comprising: 2006; Sargent et al., 2000: Savitz et al., 2009; Stockmeier, 0020 (i) introducing into the subject a positron 2003) and Selective Serotonin Reuptake Inhibitors (SSRIs) emission tomography (PET) radioligand capable of remain the first line MDD treatment, further implicating binding with a serotonin 5-HT, receptor, serotonergic dysfunction in MDD (Blier et al. 1998; G. M. 0021 (ii) performing one or more PET scans of the Sullivan et al., 2009). Tools such as Positron Emission Subject; US 2017/007 1522 A1 Mar. 16, 2017

0022 (iii) determining, by analysis of the one or 0030 (ii) performing one or more PET scans of the more PET images, a receptor binding potential of the Subject; PET radioligand for the serotonin 5-HT, receptor in 0.031 (iii) determining, by analysis of the one or more a region of interest in the Subject; PET images, a receptor binding potential of the PET 0023 (iv) comparing the receptor binding potential radioligand for the serotonin 5-HT, receptor in a value of the radioligand in the region of interest in region of interest in the Subject; the Subject to a predetermined receptor binding 0.032 (iv) comparing the receptor binding potential potential threshold value; and value of the PET radioligand in the region of interest in 0024 (V) classifying the subject as afflicted with the the Subject to a predetermined receptor binding poten depressive disorder when the receptor binding poten tial threshold value; and tial value of the radioligand in the Subject is greater 0033 (V) classifying the subject as having the depres than the predetermined diagnostic threshold value; sive disorder or as not having the depressive disorder and based on the comparison of step (iv), thereby deter mining whether the subject is afflicted with the depres 0025 (b) treating the subject based on the determina sive disorder. tion obtained in step (a). 0034. The present invention also provides a method of preparing a report classifying a subject as having a depres BRIEF DESCRIPTION OF THE FIGURES sive disorder or as not having a depressive disorder which 0026 FIG. 1: 'CIWAY-100635 binding potential (BP) comprises: estimates for the 5-HT, receptor in male control and male 0035 (i) receiving the data of one or more PET scans MDD subjects in the raphe nuclei. The horizontal dotted line of the subject performed by a PET imaging device after represents a threshold value that can separate control Sub a PET radioligand for a serotonin 5-HT, receptor was jects from MDD subjects. This threshold was used to introduced into the subject; calculate the sensitivity and specificity of diagnosis. Dia 0.036 (ii) processing the data to determine a receptor monds or squares represent single measurements of raphe binding potential of the PET radioligand for the sero BP in control and MDD subjects, respectively. Thin capped tonin 5-HT, receptor in a region of interest in the vertical error bars represent standard errors computed using Subject and comparing the receptor binding potential a bootstrap algorithm that takes into account errors in value to a predetermined receptor binding potential metabolite, plasma, and image data. Weighted group mean threshold value; and and standard error of the weighted mean of BP are repre 0037 (iii) populating a report classifying the subject. sented by thick horizontal lines and thick vertical lines, 0038. The present invention further provides a method of respectively. BPF, binding potential: 'CIWAY-100635, treating a subject afflicted with a depressive disorder, com N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2- prising pyridinyl)cyclo-hexanecarboxamide, 5-HT, serotonin-1A 0.039 (a) determining whether the subject is afflicted receptor. with the depressive disorder comprising: 0027 FIG. 2: 'CIWAY-100635 binding potential (BP) 0040 (i) introducing into the subject a positron estimates for the 5-HT, receptor in male control, male emission tomography (PET) radioligand capable of MDD, male HRO and male remitted subjects in the raphe binding with a serotonin 5-HT, receptor, nuclei. Diamonds, squares, triangles and X's represent single 0041 (ii) performing one or more PET scans of the measurements of raphe BP in control, MDD, HRO and Subject; remitted subjects, respectively. Thin capped vertical error 0042 (iii) determining, by analysis of the one or bars represent standard errors computed using a bootstrap more PET images, a receptor binding potential of the algorithm that takes into account errors in metabolite, PET radioligand for the serotonin 5-HT, receptor in plasma, and image data. Weighted group mean and standard a region of interest in the Subject; error of the weighted mean of BP are represented by thick 0043 (iv) comparing the receptor binding potential horizontal lines and thick vertical lines, respectively. BPF, binding potential; 'CIWAY-100635, N-(2-(4-(2-methoxy value of the PET radioligand in the region of interest phenyl)-1-piperazinyl)ethyl)-N-(2-pyridinyl)cyclo-hexan in the Subject to a predetermined receptor binding ecarboxamide: MDD, Major Depressive Disorder; HRO, potential threshold value; and High Risk Offspring (men who have never had depression 0044 (V) classifying the subject as afflicted with the themselves, but have at least one parent diagnosed with depressive disorder when the receptor binding poten MDD); Remitted, previously depressed men who have tial value of the PET radioligand in the subject is remitted from a major depressive episode: 5-HT, sero greater than the predetermined diagnostic threshold tonin-1A receptor. value; and 0.045 (b) treating the subject based on the determina DETAILED DESCRIPTION OF THE tion obtained in step (a). INVENTION 0046. In some embodiments, the PET radioligand is introduced by injection into the bloodstream of the subject. 0028. The present invention provides a method of deter 0047. In some embodiments, the analysis of the one or mining whether a subject is afflicted with a depressive more PET images in step (iii) is a computer analysis. disorder comprising: 0048. In some embodiments, the step (ii) further com 0029 (i) introducing into the subject a positron emis prises carrying out one or more MRI scans of the Subject. sion tomography (PET) radioligand capable of binding 0049. In some embodiments, the MRI images are ana with a serotonin 5-HT, receptor; lyzed to define the boundaries of the region of interest. US 2017/007 1522 A1 Mar. 16, 2017

0050. In some embodiments, the MRI images are ana 44.0. In some embodiments, the methods wherein the pre lyzed to define the boundaries of some of the region of determined receptor binding potential threshold value is interest. 46.0. In some embodiments, the methods wherein the pre 0051. In some embodiments, the subject is classified as determined receptor binding potential threshold value is having the depressive disorder when the receptor binding 48.0. In some embodiments, the methods wherein the pre potential value of the PET radioligand in the region of determined receptor binding potential threshold value is interest in the Subject is greater than the predetermined 5O.O. diagnostic threshold value. 0064. In some embodiments, the methods wherein the 0052. In some embodiments, the subject is classified as predetermined receptor binding potential threshold value is not having the depressive disorder when the receptor bind 39.9. ing potential value of the PET radioligand in the region of 0065. In some embodiments, the depressive disorder is interest in the subject is about the same or less than the major depression. predetermined diagnostic threshold value. 0066. In some embodiments, the subject is a human 0053. In some embodiments, the PET radioligand con Subject. tains a radioisotope selected from the group consisting of 0067. In some embodiments, the human subject is a male H, IC, 13N, 18F 123, 125I, 99mTc, 95Tc, Ill In, 62Cu, 6.Cu, Subject. Sc, 7 Ga, and Ga. 0068. In some embodiments, the human subject is a 0054. In some embodiments, the PET radioligand con female subject. tains a 'C radioisotope. In some embodiments, the PET 0069. In some embodiments, the subject had never under radioligand contains a 'F radioisotope. gone treatment. In some embodiments, the 0055. In some embodiments, the PET radioligand is Subject had gone without antidepressant treatment for at radiolabeled N-2-4-(2-methoxyphenyl)-1-piperazinyl least four years. ethyl-N-2-pyridinylcyclohexanecarboxamide. 0070. In some embodiments, the male subject had never 0056. In some embodiments, the radiolabeled N-2-4- undergone antidepressant treatment. In some embodiments, (2-methoxyphenyl)-1-piperazinylethyl-N-2-pyridinylcy the male Subject had gone without antidepressant treatment clohexanecarboxamide is radiolabeled with carbon-11. for at least four years. 0057. In some embodiments, the radiolabeled N-2-4- 0071. In some embodiments, the subject is treated with (2-methoxyphenyl)-1-piperazinylethyl-N-2-pyridinylcy an anti-depressant. In some embodiments, the male Subject clohexanecarboxamide is radiolabeled with carbon-11 at the is treated with an anti-depressant. carbonyl carbon. 0072. In some embodiments, the anti-depressant is 0.058. In some embodiments, the radiolabeled N-2-4- selected from the group consisting of , Escitalo (2-methoxyphenyl)-1-piperazinylethyl-N-2-pyridinylcy pram, , , , , Des clohexanecarboxamide is radiolabeled with carbon-11 at the , , , , methyl carbon of the methoxy group. Venlafaxine, , , , Luba 0059. In some embodiments, the region of interest is in Zodone, , , , , the brain. , , , Dextroamphetamine, 0060. In some embodiments, the region of interest is Dextromethamphetamine, LisdeXamfetamine, Amitrip selected from the group consisting of the raphe nucleus, tyline, , , , DoSule dorsolateral prefrontal cortex, medial prefrontal cortex, pin, , , , , Meli orbito-frontal cortex, anterior cingulate cortex, Subgenual tracen, , . , prefrontal cortex, temporal cortex, parietal cortex, occipital . , , , Mir cortex, amygdala, uncus, hippocampal formation, entorhinal tazapine, Isocarboxazid, Phenelzine, Selegiline, Tranylcy cortex, parahippocampal gyrus, insula, dorsal raphe nuclei. promine, Moclobemide, Pirlindole, Mianserin, , and cerebellum. , , , , and 0061. In some embodiments, the region of interest is the AZD6765. dorsal raphe nuclei. 0073. In some embodiments, the subject is treated with 0062. In some embodiments, the region of interest is the psychotherapy. In some embodiments, the male Subject is raphe nuclei. treated with psychotherapy. 0063. In some embodiments, the methods wherein the 0074. In some embodiments, the psychotherapy is predetermined receptor binding potential threshold value is selected from the group consisting of Psychodynamic 30.0. In some embodiments, the methods wherein the pre Therapy, Interpersonal Therapy and Cognitive Behavioral determined receptor binding potential threshold value is Therapy. 32.0. In some embodiments, the methods wherein the pre 0075. The present invention provides a method of deter determined receptor binding potential threshold value is mining whether a male subject is afflicted with a depressive 34.0. In some embodiments, the methods wherein the pre disorder comprising: determined receptor binding potential threshold value is 0.076 (i) introducing into the male subject a positron 36.0. In some embodiments, the methods wherein the pre emission tomography (PET) radioligand capable of determined receptor binding potential threshold value is binding with a serotonin 5-HT, receptor, 38.0. In some embodiments, the methods wherein the pre 0.077 (ii) performing one or more PET scans of the determined receptor binding potential threshold value is male Subject; 40.0. In some embodiments, the methods wherein the pre 0078 (iii) determining, by analysis of the one or more determined receptor binding potential threshold value is PET images, a receptor binding potential of the PET 42.0. In some embodiments, the methods wherein the pre radioligand for the serotonin 5-HT, receptor in a determined receptor binding potential threshold value is region of interest in the male Subject; US 2017/007 1522 A1 Mar. 16, 2017

0079 (iv) comparing the receptor binding potential binding potential value of the PET radioligand in the region value of the PET radioligand in the region of interest in of interest in male Subject is greater than the predetermined the male Subject to a predetermined receptor binding diagnostic threshold value. potential threshold value; and 0099. In some embodiments, the male subject is classi 0080 (v) classifying the male subject as having the fied as not having the depressive disorder when the receptor depressive disorder or as not having the depressive binding potential value of the radioligand in the region of disorder based on the comparison of step (iv), thereby interest in the male subject is about the same or less than the determining whether the male subject is afflicted with predetermined diagnostic threshold value. the depressive disorder. 0100. In some embodiments, the PET radioligand con 0081. The present invention also provides a method of tains a radioisotope selected from the group consisting of preparing a report classifying a male Subject as having a H, IC, 13N, 18F 123, 125I, 99mTc, 95Tc, Ill In, 62Cu, (Cu, depressive disorder or as not having a depressive disorder Sc Ga, and Ga. which comprises: 0101. In some embodiments, the PET radioligand con I0082 (i) receiving the data of one or more PET scans tains a 'C radioisotope. of the male subject performed by a PET imaging device 0102. In some embodiments, the PET radioligand con after a PET radioligand for a serotonin 5-HT, receptor tains a F radioisotope. was introduced into the male Subject; 0103) In some embodiments, the PET radioligand is I0083 (ii) processing the data to determine a receptor radiolabeled N-2-4-(2-methoxyphenyl)-1-piperazinyl binding potential of the PET radioligand for the sero ethyl-N-2-pyridinylcyclohexanecarboxamide. tonin 5-HT, receptor in a region of interest in the male 0104. In some embodiments, the radiolabeled N-2-4- Subject and comparing the receptor binding potential (2-methoxyphenyl)-1-piperazinylethyl-N-2-pyridinylcy value to a predetermined receptor binding potential clohexanecarboxamide is radiolabeled with carbon-11. threshold value; and 0105. In some embodiments, the radiolabeled N-2-4- I0084 (iii) populating a report classifying the male (2-methoxyphenyl)-1-piperazinylethyl-N-2-pyridinylcy Subject. clohexanecarboxamide is radiolabeled with carbon-11 at the 0085. The present invention further provides a method of carbonyl carbon. treating a male subject afflicted with a depressive disorder, 0106. In some embodiments, the radiolabeled N-2-4- comprising (2-methoxyphenyl)-1-piperazinylethyl-N-2-pyridinylcy I0086 (a) determining whether the male subject is clohexanecarboxamide is radiolabeled with carbon-11 at the afflicted with the depressive disorder comprising: methyl carbon of the methoxy group. I0087 (i) introducing into the male subject a positron 0107. In some embodiments, the PET radioligand is emission tomography (PET) radioligand capable of radiolabeled 2-(4-(4-(2-methoxyphenyl)piperazin-1-yl)bu binding with a serotonin 5-HT, receptor; tyl)-4-methyl-1,2,4-triazine-3.5 (2H4H) dione (''C- I0088 (ii) performing one or more PET scans of the CUMI). male Subject; 0108. In some embodiments, the radiolabeled 2-(4-(4-(2- I0089 (iii) determining, by analysis of the one or methoxyphenyl)piperazin-1-yl)butyl)-4-methyl-1,2,4-triaz more PET images, a receptor binding potential of the ine-3.5 (2H4H) dione is radiolabeled with carbon-11. PET radioligand for the serotonin 5-HT, receptor in 0109. In some embodiments, the radiolabeled 2-(4-(4-(2- a region of interest in the male Subject; methoxyphenyl)piperazin-1-yl)butyl)-4-methyl-1,2,4-triaz 0090 (iv) comparing the receptor binding potential ine-3.5 (2H4H) dione is radiolabeled with carbon-11 at the value of the PET radioligand in the region of interest methoxy carbon. in the male Subject to a predetermined receptor 0110. In some embodiments, the region of interest is in binding potential threshold value; and the brain. 0091 (V) classifying the subject as afflicted with the 0111. In some embodiments, the region of interest is depressive disorder when the receptor binding poten selected from the group consisting of the raphe nucleus, tial value of the radioligand in the male subject is dorsolateral prefrontal cortex, medial prefrontal cortex, greater than the predetermined diagnostic threshold orbito-frontal cortex, anterior cingulate cortex, Subgenual value; and prefrontal cortex, temporal cortex, parietal cortex, occipital 0092 (b) treating the male subject based on the deter cortex, amygdala, uncus, hippocampal formation, entorhinal mination obtained in step (a). cortex, parahippocampal gyrus, insula, dorsal raphe nuclei. 0093. In some embodiments, the PET radioligand is and cerebellum. introduced by injection into the bloodstream of the male 0112. In some embodiments, the region of interest is the Subject. dorsal raphe nuclei. 0094. In some embodiments, the analysis of the one or 0113. In some embodiments, the region of interest is the more PET images in step (iii) is a computer analysis. raphe nuclei. 0095. In some embodiments, step (ii) further comprises 0114. In some embodiments, the predetermined receptor carrying out one or more MRI scans of the Subject. binding potential threshold value is 30.0. In some embodi 0096. In some embodiments, the MRI images are ana ments, the methods wherein the predetermined receptor lyzed to define the boundaries of the region of interest. binding potential threshold value is 32.0. In some embodi 0097. In some embodiments, the MRI images are ana ments, the predetermined receptor binding potential thresh lyzed to define the boundaries of some of the region of old value is 34.0. In some embodiments, the predetermined interest. receptor binding potential threshold value is 36.0. In some 0098. In some embodiments, the male subject is classi embodiments, the predetermined receptor binding potential fied as having the depressive disorder when the receptor threshold value is 38.0. In some embodiments, the prede US 2017/007 1522 A1 Mar. 16, 2017 termined receptor binding potential threshold value is 40.0. 0.129 (v) classifying the subject as having the depres In some embodiments, the predetermined receptor binding sive disorder or as not having the depressive disorder potential threshold value is 42.0. In some embodiments, the based on the comparison of step (iv), thereby deter predetermined receptor binding potential threshold value is mining whether the Subject is at risk for developing a 44.0. In some embodiments, the predetermined receptor depressive disorder. binding potential threshold value is 46.0. In some embodi 0.130. The present invention provides a method of deter ments, the predetermined receptor binding potential thresh mining whether a male subject is afflicted with a depressive old value is 48.0. In some embodiments, the predetermined disorder comprising: receptor binding potential threshold value is 50.0. 0131 (i) introducing into the male subject a positron 0115. In some embodiments, the predetermined receptor emission tomography (PET) radioligand capable of binding potential threshold value is 39.9. binding with a serotonin 5-HT, receptor in the raphe 0116. In some embodiments, the depressive disorder is nuclei in the male Subject; major depression. 0.132 (ii) performing one or more PET scans of the 0117. In some embodiments, the human male subject had male Subject; never undergone antidepressant treatment. 0.133 (iii) determining, by analysis of the one or more 0118. In some embodiments, the human male subject had PET images, a receptor binding potential of the PET gone without antidepressant treatment for at least four years. radioligand for the serotonin 5-HT, receptor in the 0119. In some embodiments of the above method, the raphe nuclei in the male Subject; depressive disorder is major depression. 0.134 (iv) comparing the receptor binding potential 0120 In some embodiments of the above method, the value of the radioligand in the raphe nuclei in the male male Subject is treated with an anti-depressant. Subject to a predetermined receptor binding potential 0121. In some embodiments of the above method, the threshold value; and male subject is treated with selective serotonin reuptake 0.135 (v) classifying the male subject as having the inhibitors (SSRIs), serotonin and norepinephrine reuptake depressive disorder or as not having the depressive inhibitors (SNRIs), atypical , tricyclic anti disorder based on the comparison of step (iv), thereby depressants, tetracyclic antidepressants, or monoamine oxi determining whether the male subject is afflicted with dase inhibitors (MAOIs). the depressive disorder. 0122. In some embodiments of the above method, the 0.136. In some embodiments of the above method, the anti-depressant is selected from the group consisting of predetermined receptor binding potential threshold value Citalopram, , Paroxetine, Fluoxetine, Fluvox radioligand in the raphe nuclei is 30.0, 32.0, 34.0, 36.0, 38.0, amine, Sertraline, , Duloxetine, Levomil 40.0, 42.0, 44.0, 46.0, 48.0, or 50.0. nacipran, Milnacipran, Venlafaxine, Tratnadol, Sibutramine, 0.137 In some embodiments of the above method, the Etoperidone, Lubazodone, Nefazodone, Trazodone, Atom predetermined receptor binding potential threshold value oxetine, Reboxetine, Viloxazine, Bupropion, Amphetamine, radioligand in the raphe nuclei is 39.9. Dextroamphetamine, Dextromethamphetamine, Lisdexam 0.138. The present invention provides a method of deter fetamine, , Butriptyline, Clomipramine, Desip mining whether a subject is at risk for developing a depres ramine, , Doxepin, Imipramine, prindole, Lofe sive disorder comprising: pramine, , Nortriptyline, Opipramol. 0.139 (i) introducing into the subject a positron emis Protriptyline, Trimipramine, Amoxapine, Maprotiline, sion tomography (PET) radioligand capable of binding Mianserin, Mirtazapine, Isocarboxazid, Phenelzine, Selegi with a serotonin 5-HT, receptor in the raphe nuclei of line, Tranylcypromine, Moclobemide, Pirlindole, Mianserin, the subject; Mirtazapine, Vilazodone, Vortioxetine, Tandospirone, Que 0140 (ii) performing one or more PET scans of the tiapine, and AZD6765. Subject; 0123. In some embodiments of the above method, the 0.141 (iii) determining, by analysis of the one or more male Subject is treated with psychotherapy. In some embodi PET images, a receptor binding potential of the PET ments, the psychotherapy is selected from the group con radioligand for the serotonin 5-HT, receptor in the sisting of Psychodynamic Therapy, Interpersonal Therapy raphe nuclei in the Subject; and Cognitive Behavioral Therapy. 0.142 (iv) comparing the receptor binding potential 0.124. The present invention provides a method of deter value of the radioligand in the subject to a predeter mining whether a Subject is at risk for developing a depres mined receptor binding potential threshold value; and sive disorder comprising: 0.143 (V) classifying the subject as at risk for devel 0.125 (i) introducing into the subject a positron emis oping the depressive disorder or as not at risk for sion tomography (PET) radioligand capable of binding developing the depressive disorder based on the com with a serotonin 5-HT, receptor; parison of step (iv), thereby determining whether the 0.126 (ii) performing one or more PET scans of the subject is at risk for developing the depressive disorder. Subject; 0144. The present invention provides a method of deter I0127 (iii) determining, by analysis of the one or more mining whether a male Subject is at risk for developing a PET images, a receptor binding potential of the PET depressive disorder comprising: radioligand for the serotonin 5-HT, receptor in a 0145 (i) introducing into the male subject a positron region of interest in the Subject; emission tomography (PET) radioligand capable of I0128 (iv) comparing the receptor binding potential binding with a serotonin 5-HT, receptor in the raphe value of the PET radioligand in the region of interest in nuclei of the male subject; the Subject to a predetermined receptor binding poten 0146 (ii) performing one or more PET scans of the tial threshold value; and male Subject; US 2017/007 1522 A1 Mar. 16, 2017

0147 (iii) determining, by analysis of the one or more PET images, a receptor binding potential of the PET radioligand for the serotonin 5-HT, receptor in the raphe nuclei of the male subject; 0148 (iv) comparing the receptor binding potential value of the radioligand in the male Subject to a predetermined receptor binding potential threshold --O value; and N 0149 (v) classifying the male subject as at risk for --- developing the depressive disorder or as not at risk for developing the depressive disorder based on the com 0159. In some embodiments, BPF, including raphe BPF, parison of step (iv), thereby determining whether the is calculated using the tracer 'CCUMI-101, a 5-HT male Subject is at risk for developing the depressive partial agonist. ''CCUMI-101 allows for the BPF to be disorder. calculated without the need for blood sampling and the insertion of an arterial cannula (Hendry, N. et al., 2011; 0150. In some embodiments of the above method, the Milak, M. S. et al., 2008; Milak, M. S. et al., 2010b). predetermined receptor binding potential threshold value (0160. In some embodiments, the tracer is O-methyl-'C) radioligand in the raphe nuclei is 30.0, 32.0, 34.0, 36.0, 38.0, 2-(4-(4-(2-methoxyphenyl)piperazin-1-yl)butyl)-4-methyl 40.0, 42.0, 44.0, 46.0, 48.0, or 50.0. 1,2,4-triazine-3.5 (2H4H) dione (''C-CUMI). 0.161. In some embodiments, BPF, including raphe BP, 0151. In some embodiments of the above method, the is calculated using an 'F labeled CUMI-101. Due to its predetermined receptor binding potential threshold value 109-minute half-life, an F-18 version of this PET tracer can radioligand in the raphe nuclei is 39.9. be shipped to other research centers and does not require a 0152. In some embodiments of any of the above methods, cyclotron on site in order to make the radiotracer. the Subject is a human male Subject. 0162. As used herein, “Receptor Binding Potential”, “Binding Potential” or “BPF refers to the ratio at equilib 0153. In some embodiments of any of the above methods, rium of the concentration of specifically bound radioligand the subject is a human female subject. in tissue to the concentration of free radioligand in tissue. 0163 Binding potential in a region of interest refers to 0154) In some embodiments of any of the above methods, the ratio at equilibrium of the concentration of specifically the depressive disorder is major depression. bound radioligand in tissue of the region of interest to the 0155. In some embodiments of any of the above methods, concentration of free radioligand in tissue (Innis et al., 2007). the one or more PET scans or one or more MRI scans are 0164. In some embodiments, the radioligand binds to performed on the region of interest in the Subject. serotonin 5-HT, receptors in the tissue of the region of interest. 0156 N-2-[4-(2-Methoxyphenyl)-1-piperazinylethyl 0.165. As used herein, “predetermined receptor binding N-2-pyridinylcyclohexanecarboxamide (WAY-100635) has potential threshold value' refers to a threshold value of the following structure and is available for purchase as receptor binding potential of a PET radioligand to serotonin Catalog No. W108 from Sigma-Aldrich (St. Louis, Mo., 5-HT, receptors in a region of interest. USA): 0166 In some embodiments, the “predetermined receptor binding potential threshold value' refers to a threshold value of receptor binding potential of a PET radioligand to sero tonin 5-HT, receptors in the raphe (the region from which all serotonergic neurons originate). This threshold is indi cated by the green dotted line in FIG. 1. 0167. In order to determine the proper threshold for N O diagnostic classification, the diagnostic sensitivity and specificity associated with using each measured raphe BP No r^- N value as the diagnostic cutoff were calculated. The minimum Nu N,N-4 value of a cost function consisting of the (negative) sum of these sensitivity and specificity measures was sought. The threshold of 39.9 mL/cm is a solution that minimized this cost function. Due to the separation of subjects at this level of binding, the threshold can be determined to within 3 mL/cm. 0.168. In some embodiments, the predetermined receptor binding potential threshold value is determined by analyzing 0157. The preparation of Carbonyl-'CIWAY-100635 is a male control Subject or group of male control Subjects that described in Hwang et al., 1999, the contents of which is are not afflicted with a depressive disorder. hereby incorporated by reference. 0169. In some embodiments, the predetermined receptor 0158 2-(4-(4-(2-methoxyphenyl)piperazin-1-yl)butyl)- binding potential threshold value is determined by analyzing 4-methyl-1,2,4-triazine-3.5 (2H4H) dione (CUMI-101) has a male control Subject or group of male control Subjects that the following structure: have not been diagnosed with a depressive disorder. US 2017/007 1522 A1 Mar. 16, 2017

0170 In some embodiments, the predetermined receptor Clinical Assessments binding potential threshold value is determined by analyzing a male control Subject or group of male control Subjects that (0176) The HDRS-17 (Hamilton, 1960), the Beck Inven have been diagnosed as not having the depressive disorder. tory (BDI) (Beck et al., 1961), and the Global Assessment Scale (Endicott et al., 1976) were utilized to assess depres (0171 In some embodiments, the free fraction (f) of a sion severity and functional impairment. PET tracer, measured from a single venous sample is used in the calculation of BP (Milak, M.S. et al., 2010a). The f, value for Carbonyl-'CIWAY-100635 is directly correlated Radiochemistry and Input Function Measures with 5-HT, BP in the raphe nucleus (RN) and Carbonyl 0177. Measurements of arterial input function, metabo ''CIWAY-100635 acts as a surrogate biomarker for MDD. lites and free plasma fraction (f) were also made as With a direct correlation present, simple venous sampling described previously (Parsey et al., 2005; Parsey et al., following administration of Carbonyl-'CIWAY-100635 2000). allows for MDD diagnosis. Genotyping 0172. In the present application, all numbers disclosed herein may vary by 1 percent, 2 percent, 5 percent, or up to 0.178 The functional 5-HT gene promoter region single 20 percent if the word “about is used in connection there nucleotide polymorphism (SNP) known as 5-HT with. This variation may be applied to all numbers disclosed C(-1019)G was genotyped for a bi-allelic classification i.e. herein. CC, CG or GG, for each participant as previously described 0173 Each embodiment disclosed herein is contemplated (Huang et al., 2004: Wu & Comings, 1999). as being applicable to each of the other disclosed embodi ments. Thus, all combinations of the various elements PET Acquisition described herein are within the scope of the invention. 0179 Imaging was performed as described Parsey et al., (0174. This invention will be better understood by refer 2000, the contents of which is hereby incorporated by ence to the Experimental Details which follow, but those reference. Briefly, after an Allen test and subcutaneous skilled in the art will readily appreciate that the specific administration of 2% lidocaine, a catheter was inserted in the experiments detailed are only illustrative of the invention as radial artery. A venous catheter was also inserted into a described more fully in the claims which follow thereafter. forearm vein on the opposite side for PET tracer adminis tration. Head movement was minimized with a polyurethane Experimental Details head immobilizer system (Soule Medical, Tampa, Fla., USA), molded around the head of the subject. PET imaging was performed with the ECAT EXACT HR+(Siemens/CTI, Materials and Methods Knoxville, Tenn., USA) (63 slices covering an axial field of view of 15.5 cm, axial sampling of 2.46 mm, in 3D mode. Participants A 10-min transmission scan was acquired before injection. 0.175. This study was approved by the Institutional After injection of 'CIWAY-100635, as an intravenous Review Boards of the New York State Psychiatric Institute bolus over 45 secs using an injection pump, emission data and Columbia University Medical Center. 107 subjects were collected for 110 mins as 20 successive frames of between the ages of 18 and 65 were evaluated in this study. increasing duration (3x20 secs, 3x1 min, 3x2 mins, 2x5 These subjects were a combined cohort from three previ mins, 9x10 mins). Images were reconstructed using the ously reported studies (Miller et al., 2009; Parsey et al., 3D-RP algorithm implemented on a vector processor (CTI, 2010; Parsey et al., 2006) as well as 11 additional subjects. Knoxville, Tenn., USA) to a 128x128 matrix (pixel size of Fifty subjects met DSM (Diagnostic and Statistical Manual 1.7x1.7 mm) with attenuation correction and a Shepp 0.5 of Mental Disorders) IV criteria for Major Depressive Dis filter (cutoff 0.5 cycles/projection rays) resulting in an order (34 female, 16 male) and fifty-seven were healthy in-plane and axial resolution (i.e. full width half-maximum) volunteers (32 female, 25 male). All participants provided of 4.4 mm and 4.1 mm in air and at the center of the field written informed consent after learning the description of the of view (Brix et al., 1997). Scatter correction was performed study protocol. All diagnoses were agreed upon by at least using the technique implemented by the manufacturer (Wat three senior psychiatrists. All of the MDD subjects were son et al., 1995). categorized as Not Recently Medicated (NRM): defined as greater than 4 years since antidepressant treatment. Study Input Function Measurement criteria for depressed subjects included: 1) age 18 to 65 0180 Input function and measurement of metabolites years; 2) DSM IV criteria for current MDD; 3) absence of were conducted as described previously (Parsey et al., any psychotropic for at least 2 weeks (4 years 2000). Briefly, after radiotracer injection, 30 arterial samples for antidepressants, 4 weeks for neuroleptics), except ben were collected every 5 secs with an automated Sampling Zodiazepines, which were discontinued three days prior to system for the first 2 mins, and manually thereafter at longer the scan; 4) absence of lifetime history of or sub intervals. After centrifugation (10 mins at 3800 g), plasma stance abuse or dependence; 5) absence of life-time expo was collected in 200-4 aliquots and radioactivity was sure to 3.4-methylenedioxymethamphetamine (MDMA, counted in a gamma counter (Wallac 1480 Wizard 3 M “ecstasy'); 6) absence of significant medical conditions; 7) Automatic Gamma Counter). Five samples were processed absence of pregnancy; 8) capacity to provide informed to measure the fraction of unmetabolized 'CIWAY-100635 consent; and 9) absence of psychosis, bipolar disorder, or by high-pressure liquid chromatography (HPLC). The five Schizophrenia. Screening was performed via physical exam, measured unmetabolized 'CIWAY-100635 fractions were history, routine blood and urine tests. fit with the Hill function (Gunn et al., 1998). The input US 2017/007 1522 A1 Mar. 16, 2017 function was the product of total counts and interpolated were generated by plotting the average regional activity unmetabolized 'CIWAY-100635 fraction. The measured within each co-registered PET frame over the time of the input fountain values (CCt), uCi/mL) were fit to a straight SCall. line from time zero to the peak followed by the sum of three exponentials after the peak. The fitted values were used as Quantitative Analysis input to the kinetic analysis. For the determination of the plasma free fraction (f), triplicate 200-uL aliquots of 0183) Regional distribution volumes of 'CIWAY plasma collected before injection were mixed with a 100635 were derived from kinetic analysis using the arterial radiotracer, pipetted into ultrafiltration units (Centrifree, input function and a two-tissue compartment (2T) model Amicon, Danvers, Mass., USA) and centrifuged at room with constrained parameters—K/K ratio fixed to that of the temperature (20 mins at 3800 g). Plasma and ultrafiltrate cerebellar white matter (see Parsey et al., 2000 for details). activities were then counted, and f, was calculated as the 0.184 The model included the concentration of tracer in ratio of ultrafiltrate to total activity concentrations (Cleare the arterial plasma compartment (C), free in tissue water and Bond, 2000). (C), nonspecifically bound (Cs), and specifically bound compartment (Cs). MRI Acquisition and Analysis 0185. The equilibrium distribution volume of a compart 0181 MRIs were acquired either on a GE 1.5 T or 3.0T ment i (V, mL/g) was defined as the ratio of the total tracer Signa Advantage system. A Sagittal Scout (localizer) was concentration in this compartment to the free plasma con performed to identify the AC-PC plane (1 min). Regions of centration at equilibrium (C/C), where C, C+C+C. interests (ROIs) were labeled on each subject’s skull 0186 V is defined as the distribution volume of the stripped and segmented (into grey/white matter and cere nondisplaceable compartment. BP (V-V)/T is equal brospinal fluid) MRI as previously described (Parsey et al., to the ratio of the available receptor density (B. nmol/L 2000) and included the ventral prefrontal cortex (VPFC), per g of tissue) and affinity (1/K nmol/L per mL of brain medial prefrontal cortex (MPFC), dorsolateral prefrontal water) (Innis et al., 2007). cortex (DLPFC), anterior cingulate cortex (ACN), cingulate 0187. The contribution of plasma total activity to the (posterior) cortex (CIN), amygdala (AMY), hippocampus regional activity was calculated assuming a 5% blood Vol (HIP), parahippocampal gyrus (PHG), insular cortex (INS), ume in the ROI and subtracted from the regional activity temporal cortex (TEM), parietal cortex (PAR), and occipital before analysis. All kinetic parameters were derived by cortex (OCC). Because the boundaries of the median and nonlinear regression using a Levenberg-Marquart least dorsal raphe nuclei (RN) are not identifiable on MRI, a 2 squares minimization procedure implemented in MATLAB cm ellipsoid was manually placed on the raphe nuclei of (The Math Works, Inc., South Natick, Mass., USA). Given each individual’s mean PET image, completely encompass the unequal sampling over time (increasing frame acquisi ing the high 'CIWAY-100635 binding region of the pos tion time from beginning to end of the study), the least terior midbrain. The reference region, cerebellar white mat squares minimization procedure was weighted by the square ter, is a region of cerebellum is virtually devoid of 5-HT root of the frame acquisition time. (Parsey et al., 2005). It was either defined from the auto mated white matter segmentation in the cerebellum or by manually outlining a circular region on the MRI. For cortical Statistics regions, the ROIs were modified, as previously described, to 0188 Standard errors (SE) were computed for each esti include only gray matter Voxels. mated BP value using a bootstrap algorithm that takes into account errors in metabolite, plasma and brain data (Ogden PET Analysis & Tarpey, 2006). ROI-level BP estimates were natural-log transformed before statistical modeling in order to account 0182 PET data analyses were performed as described for heterogeneity of variances across regions. Linear mixed previously (Parsey et al., 2005). Image analysis was per effects models with standard errors of transformed ROI formed using MATLAB (The Mathworks, Natick, Mass.) level BP estimates as weights were fit to the transformed with extensions to the following open source packages: ROI-level BP, estimates with brain region as the fixed effect Functional Magnetic Resonance Imaging of the Brain's and the subject as the random effect. The dependency Linear Image Registration Tool (FLIRT) v5. (Oxford Center structure for all ROI within the same subject was chosen for Functional Magnetic Resonance Imaging of the Brain, based on Akaike Information Criterion (AIC). The final Oxford, England) (Jenkinson & Smith, 2001), Brain Extrac structure used had a generalized compound symmetry struc tion Tool (BET) v1.2 (Oxford Centre for Functional Mag ture allowing different variance components in different netic Resonance Imaging of the Brain) (Smith, 2002), and brain regions and fixed correlation in any two brain regions University College of London's Statistical Parametric Map within the same subject. The covariates in the model ping (SPM5) (Wellcome Department of Imaging Neurosci included brain region, gender, diagnosis group, and the ence, London, United Kingdom) normalization (Ashburner interaction between brain region and gender. There were no & Friston, 1999) and segmentation routines (Ashburner & other interaction terms in first or higher order that reached Friston, 2005). To correct for subjection motion during the statistical significance level. Significance level was set at PET scan, de-noising filter techniques were applied to later 0.05 and p-values were reported without multiple compari PET images. The eighth frame was used as a reference onto son adjustment. All tests were two-sided. Model fitting was which all other frames were aligned using rigid body FLIRT. computed using both SAS 9.2 (SAS Inc., Cary, N.C.) and R For co-registration, a mean of the motion-corrected frames 3.0.2 (R Project for Statistical Computing: www.R-project. was registered using FLIRT to the MRI. Time activity curves org). US 2017/007 1522 A1 Mar. 16, 2017

Example 1 ficity=75%). However, measuring 5-HT binding in females while adjust for covariates Such as location of Effects of Sex female subjects in their menstrual cycle is useful for the 0189 Consistent with previous studies (Parsey et al., diagnosis of depressive disorders in female Subjects. Using 2006), in controls, males had a 17.8% lower BP than venous sampling, fluctuations in estrogen, and progesterone, females (df-103, p=0.0194) across all ROIs. However, in levels that occur during the menstrual cycle are taken in depressed subjects, males had a 14.7% higher BP than account to more accurately calculate measures of 5-HT females (df-103, p=0.1481). binding. 0190. When looking at each sex individually, female 0194 Female gonadal hormone levels throughout the MDD subjects had 19.7% higher BP across all regions, menstrual cycle directly affect radioligand binding to the compared to female controls (df-103, p=0.0197). As serotonin 5-HT, receptor. Binding potentials of female depicted in FIG. 1, Male MDD subjects had 67.0% higher MDD subjects and control subject obtained at a specific BP, across all regions, compared to male controls (df=103, stage of the menstrual cycle provide a greater separation p<0.0001). between diagnostic groups and higher sensitivity and speci ficity in distinguishing MDD in females. Therefore, com Example 2 parison of binding potentials obtained at a specific stage of the menstrual cycle is useful for the diagnosis of depressive Raphe Nuclei disorders in female Subjects. 0191 Post hoc analysis assessing BP, differences Example 3 between control and MDD subjects, in each sex, showed that a region by diagnosis interaction was present in each sex and Diagnosis of Depression in Male Subject that certain regions exhibit greater BP, separation between controls and MDD subjects than others (Table 1). 0.195 A PET radioligand was injected into the blood 0.192 As noted in Table 1, the largest separation in stream of a male subject. One or more PET scans were binding between MDD and control subjects occurs in the performed on the male subject. The PET images were raphe of the males. Due to this significant separation (13.2%, analyzed and a receptor binding potential of the PET radio p=0.000), this region was examined to determine a diagnos ligand at serotonin 5-HT, receptors in the raphe nuclei of tic threshold that separated male MDD and control subjects. the male subject was determined. The receptor binding In order to determine the proper threshold for diagnostic potential was determined to be greater than 39.9 and the classification, the sensitivity and specificity associated with male Subject was classified as having major depression. using each measured raphe BP value as the diagnostic 0196. A PET radioligand was injected into the blood cutoff were calculated. The minimum value of a cost func stream of a male subject. One or more PET scans were tion consisting of the (negative) sum of these sensitivity and performed the male subject. The PET images were analyzed specificity measures was sought. The threshold of 39.9 and a receptor binding potential of the PET radioligand at mL/cm is a solution that minimized this cost function. Due serotonin 5-HT, receptors in the raphe nuclei of the male to the separation of subjects at this level of binding, the Subject was determined. The receptor binding potential was threshold can be determined to within 3 mL/cm. Using this determined to be about the same or less than 39.9 and the threshold, the diagnostic sensitivity is 87.5%, specificity is male Subject was classified as not having major depression. 96.0% with a positive predictive value of 93.3% and a negative predictive value of 92.3%. Using Carbonyl-C-11 Example 4 WAY-100635 f values only achieves 56% specificity. Risk for Developing Depression TABLE 1. (0.197 Based on the data presented in FIG. 2, the threshold used for distinguishing male depressed from controls (raphe Percent Differences in BPE. Between Control and MDD Subiects BP-39 mL/cm) also distinguishes male high risk offspring (HRO) from controls. HRO offspring are individuals who Male Female have at least one parent diagnosed with depression. There RN 132.59% 10.68% fore this technology may be used to identify those at risk, AMY 64.07% 25.03% while they are currently asymptomatic. HIP 74.22% 19.59% PIP 87.96% 34.76% 0198 While the best method to separate depressed from TEM 87.07% 27.23% controls is currently based on binding potential, it is possible ACN 80.47% 30.75% that Surrogate markers are available. For example, using just CIN 80.79% 26.65% DOR 83.69% 31.10% the free fraction only to separate male MDD from male MED 84.79% 31.13% controls with a specificity of 56% or higher. Free fraction ORB 84.94% 28.59% measurement or other Such surrogate markers also identify INS 76.78% 22.48% those at risk and those currently depressed. OCC 95.18% 20.50% 0199 A PET radioligand is injected into the bloodstream PAR 98.55% 23.82% of a male subject. One or more PET scans are performed on the male subject. The PET images are analyzed and a 0193 Although, on average, the female MDD subjects receptor binding potential of the PET radioligand at sero have higher binding than female controls, the separation tonin 5-HT, receptors in the raphe nuclei of the male between diagnostic groups is much smaller (10.7%, p=0. Subject is determined. The receptor binding potential is 078), and therefore sensitivity and specificity in distinguish determined to be greater than 39.9 and the male subject is ing MDD in females is not as high (sensitivity=56%, speci classified as at risk for developing major depression US 2017/007 1522 A1 Mar. 16, 2017

0200 A PET radioligand is injected into the bloodstream human studies (though the majority of animal studies look of a male subject. One or more PET scans are performed on ing at the involvement of 5-HT, in MDD have used animal the male subject. The PET images are analyzed and a cohorts consisting of males only or using a sample with a receptor binding potential of the PET radioligand at sero disproportionate ratio of male to female subjects (Castro et tonin 5-HT, receptors in the raphe nuclei of the male al., 2003; Le Poul et al., 2000; Nishi et al., 2009)). Female Subject is determined. The receptor binding potential is ovarian sex hormones, estrogen and progesterone, have been determined to be about the same or less than 39.9 and the shown to be connected to modulation of mood (Pecins male Subject is classified as not at risk for developing major Thompson & Bethea, 1999). Further, a study looking at the depression. effect of tricyclic antidepressants (TCAS) in depressed rats exposed to chronic stress showed that TCA modulation of Discussion 5-HT mRNA transcription occurred in a sex dependent 0201 Using the PET radioligand N-(2-(4-(2-methoxy manner. Although TCA administration modulated 5-HT phenyl)-1-piperazinyl) ethyl)-N-(2-pyridinyl)cyclo-hexan mRNA expression in the cornu ammonis 1 (CA1) Sub-region ecarboxamide (carbonyl-'C-WAY-100635), a selective of the hippocampus, with hippocampal 5-HT modulation 5-HT, antagonist, it was previously shown that there are thought to be involved in MDD, positive TCA effects were higher 5-HT binding potential (BP) in MDD subjects only seen in the male rats (Pitychoutis et al., 2012). compared to control Subjects across several key regions of 0204. In humans, the decline in these sex steroids that interest (ROIs) (Parsey et al., 2006; Sullivan et al., 2009). occur in females during childbirth and menopause have been Further, it has been shown that there are differences in the correlated with negative affects including depression (Gitlin serotonergic system based on sex. In vivo, the mean rate of & Pasnau, 1989), with hormone replacement therapy (e.g. serotonin synthesis in males was estimated to be 52% higher transdermal estrogen) alleviating the depression in Some of than in females (Nishizawa et al., 1997). And, postmortem these subjects (Gregoire et al., 1996). Due to evidence such human studies have revealed sex differences in serotonin as this, one common hypothesis put forth by several groups metabolite 5-hydroxyindole-3-acetic acid (5-HIAA) levels is that depression following female sex hormone decreases (Gottfries et al., 1974). Using 'CIWAY-100635, it was is due to changes in the serotonin System within the brain reported that healthy control women had higher 5-HT causes by the changes in sex hormone levels (Eriksson et al., receptor binding compared to men (Parsey et al., 2002). In 1995: Parry et al., 1993; Halbreich & Tworek, 1997: Su et 2008, a similar study in controls found that compared to al. 1997). men, women had significantly higher 5-HT, receptor bind 0205. It then follows that sex hormones should affect ing potentials in various cortical and Subcortical regions SSRI response, which occurs through the raphe 5-HT (Jovanovic et al., 2008). 5-HT, receptor binding in each sex autoreceptor. These autoreceptors act to inhibit 5-HT post were examined separately using Subjects from previously synaptic neuronal release (Sprouse & Aghajanian. 19S6). published cohorts (Miller et al., 2009; Parsey et al., 2010; Sustained administration of 5-HT agonists or SSRIs Parsey et al., 2006) to determine whether it would provide induces the internalization of 5-HT, autoreceptors in the more insight into MDD pathophysiology and potentially raphe nucleus of the midbrain, but not of the post-synaptic give rise to a biomarker of the illness. hetero-receptors found in the hippocampus (Banerjee et al., 0202 Both animal and human data show that there are 2007). Once this receptor internalization occurs, the efficacy Substantial differences in the serotonergic system, and of SSRIs is thought to lie in the fact that the subsequent lack 5-HT modulation, between the sexes (Jovanovic et al., of autoreceptor inhibition allows increased serotonin to be 2008). As early as 1970, it was shown that central serotonin released and bind post-synaptically to 5-HT, hetero-recep levels as well as cerebrospinal fluid concentrations of the tors, therefore inducing the anxiolytic and antidepressant serotonin metabolite 5-hydroxyindole-3-acetic acid effects of SSRIs (Banerjee et al., 2007). (5-HIAA) were higher in female than male rats. Further, 0206 To date, few studies have looked at the role of sex animal literature has shown that 5-HT binding is modu in the modulation of 5-HT specifically in MDD using PET lated by estrogen levels in females (Flugge et al., 1999; in humans. However, a 2010 study showed that serotonergic Frankfurt et al., 1994; Maswood, 1995; Pecins-Thompson & differences exist in both healthy individuals and those with Bethea, 1999; Zhang et al., 1999) and that 5-HT, autore MDD across sexes. This study examined alpha-(11)C ceptors in the dorsal raphe nucleus are negatively regulated MTrp brain trapping, which is an index of serotonin Syn by female sex hormones (Birzniece et al., 2001; Maswood, thesis (Frey et al., 2010). Sex differences in serotonin 1995; Pecins-Thompson & Bethea, 1999). Conversely, rat synthesis were seen in multiple regions of the prefrontal studies have shown that estrogen induces up-regulation of cortex and limbic system, which are involved in mood 5-HT, receptors in forebrain regions such as the medial regulation. Another study looking into sex differences within preoptic area (Frankfurt et al., 1994). Finally, it was seen that the serotonergic system showed that although healthy along with female sex hormones, androgens Such as testos females exhibit lower cortical trapping of alpha-(11)C terone are able to increase the firing activity of 5-HT neurons MTrp than healthy males, females with MDD exhibit higher in both male and female rats (Robichaud & Debonnel, alpha-(11)CMTrp than males with MDD (Frey et al., 2010; 2005). Sakai et al., 2006). The current study found a similar trend 0203 The role of serotonin in depression and, in particu in that while healthy i.e. control females exhibit higher lar, the relationship between raphe 5-HT, and MDD (Kishi 5-HT binding than healthy males, females with MDD et al., 2013; Miller et al., 2009; Miller et al., 2013: Parsey et exhibit lower 5-HT binding than males with MDD. al., 2010; Parsey et al., 2006: Stockmeier et al., 1998), 0207. Several additional human PET studies have shown suggests that differences in MDD pathophysiology between that individuals with Bipolar Depression (BD) and MDD the sexes may be due to sex hormones and their modulation exhibit higher 5-HT, BP, using the radioligand carbonyl of the 5-HT, receptor. This is confirmed by both animal and C-11-WAY-100635 (Parsey et al., 2006; G. M. Sullivan et US 2017/007 1522 A1 Mar. 16, 2017

al., 2009), and that healthy females exhibit higher 5-HT with a simple venous sampling. However, it was found that BP, than men. Furthermore, post-hoc analysis in the BD Carbonyl-C-11WAY-100635 f, values achieve much lower study reported that among the male Subjects both the main specificity than BP in the raphe (56%). effect of diagnosis and the region by diagnosis interaction 0212. It is not unreasonable to use PET for depression terms were statistically significant. screening/diagnosis since, identifying those who have 0208. A possible implication of this study's findings is MDD, or are at risk for MDD, as early as possible will save that previously published reports on the separation of MDD time and money in diagnosis and early intervention. How and healthy controls, based on 5-HT binding, were poten ever, if this technique were extended to clinical use, it would tially driven by the males (Parsey et al., 2010; Parsey et al., be greatly beneficial to use additional 5-HT PET tracers. 2006). However, this is not to suggest that there are no 0213 Raphe BP can be calculated using the tracer differences between control and MDD subjects within the 'CCUMI-101, a 5-HT, partial agonist. This tracer allows female cohort. Rather, similar to the previous preclinical and for the BP estimates to be calculated without the need for clinical studies cited above, the results suggest that the blood sampling and the insertion of an arterial cannula pathogenesis of MDD between the two sexes may be (Hendry, 2011; Milak, 2008, 2010b). An F-18 version of different, although the prevalence may be equal when 'CCUMI-101 is also used as a tracer. Due to its 109 accounting for the different ways in which men and women minute half-life, an F-18 version of this PET tracer can be experience depression. shipped to other research centers and does not require a 5-HT, RN BP, as a Biomarker for MDD Diagnosis cyclotron on site in order to make the radiotracer. In contrast, 0209. Using neuroimaging, several possible endopheno compounds made with C-11 have a half-life of only 20 types for several psychopathologies have been proposed and minutes. include: amygdala/hippocampal Volumes in borderline per sonality disorder via the use of (Ruocco, 2012), brain and SUMMARY CSF volumes in Alzheimer's disease (Reitz, 2009), and 0214 Currently, there are no diagnostic tests one can white matter pathology and brain volumetric differences in perform to determine if an individual is clinically depressed. bipolar disorder (Borgwardt, 2012; Hajek, 2005). There The clinician must make this determination based on the even exist multiple potential non-imaging based markers for patient's self-report and the clinician’s judgment. This Sub diagnosing MDD i.e. growth factors, cytokines and endo jective system is prone to error and is also unrelated to the crine factors, however these markers are limited by a lack of biological causes of depression. sensitivity and specificity and have not be translated into (0215 Using 5-HT, BP in the raphe as a biomarker for clinical practice. MDD is an extremely promising concept for the field of 0210. This study found that using a threshold value to psychiatry. Although PET technology is currently expensive, categorize subjects as either MDD or control based solely on it is still used millions of times each year in a variety of raphe 5-HT, BP, values yielded extremely high diagnostic medical conditions. Being able to identify those who have sensitivity and specificity (87.5% and 96%, respectively). MDD, or are at risk for MDD, as early as possible saves Using elevated 5-HT, as a biomarker or endophenotype of downstream time and money in diagnosis and early inter MDD could significantly advance our understanding of this vention. psychopathology in several ways. As pointed out by Peter 0216) Positron Emission Tomography (PET) is a tech son et al (Peterson, 2011), a biomarker for MDD could aid: nique used to visualize and quantify targets in the brain Such in classifying the great heterogeneity observed across MDD as neuroreceptors. It is a tool that can be used to understand presentation into identifiable Sub-diagnostic categories and the disruption of neurotransmitter systems that may occur in therefore allow more customized treatment strategies; the depression and other neurological and psychiatric disorders. search for genetic and environmental factors; in identifying The systems visualized by PET are dependent on the those likely to have a chronic course, be treatment resistant, radiotracer used, and the accuracy of the PET quantification or respond to vs. therapy; and in identifying is dependent on the techniques used to analyze the resulting those at increased risk for MDD. The last possibility is especially important since MDD is only 31-42% genetically PET images. 0217 Serotonin 5-HT binding was found to be higher determined. For this reason, clinicians cannot accurately in healthy females than in healthy males. However, in predict who will develop the illness based only on family depressed subjects, serotonin 5-HT binding was higher in history (P. F. Sullivan et al., 2000). Being able to quantify the males than females. Further analysis focused on sero their risk of developing MDD, would allow for preventative tonin 5-HT binding in the raphe, a small region in the strategies to be taken to improve their future mental health midbrain from which most serotonergic neurons originate, outcomes. The current MDD diagnostic criteria do not in males. Raphe BP, reveals significant differences between provide insight into these questions to the extent that a male depressed (higher binding) and control Subjects. In biomarker would. fact, if a diagnostic threshold of BP, 39.9 mL/cm is used, 0211 For these reasons, using 5-HT, BP, in the raphe as male depressed and control Subjects can be distinguished a biomarker for MDD is an extremely promising concept. with 96% specificity and 87.5% sensitivity. As such, this However, one confound to the practicality of PET in clinical method could be the first objective diagnostic test for psychiatry is the invasiveness of the procedure i.e. the need clinical depression. of arterial cannulas to calculate BP binding measures. The free fraction (f) of a PET tracer, measured from a single REFERENCES venous sample (Milak, 2010a) is used in the calculation of BPF. If the f, value for Carbonyl-C-11WAY-100635 was 0218. Ashbumer, J., & Friston, K. J. (1999). Nonlinear directly correlated with 5-HT, BP, in the RN, it could be spatial normalization using basis functions. Hum Brain surrogate biomarker for BPF, allowing diagnosis of MDD Mapp. 7(4), 254-266. US 2017/007 1522 A1 Mar. 16, 2017

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0286 Sullivan, P. F. et al. (2000). Genetic epidemiology 11. The method of claim 9, wherein the PET radioligand of major depression: review and meta-analysis. Am J is radiolabeled N-(2-4-(2-methoxyphenyl)-1-piperazinyl Psychiatry, 157, 1552-1562. ethyl-N-2-pyridinylcyclohexanecarboxamide or radiola (0287 Woods, S. W. (2000). The economic burden of beled 2-(4-(4-(2-methoxyphenyl)piperazin-1-yl)butyl)-4- bipolar disease. J Clin Psychiatry, 61 Supp. 13, 38-41. methyl-1,2,4-triazine-3.5 (2H4H)dione. 0288 Wu, S., & Comings, D. E. (1999). A common 12.-17. (canceled) C-1018G polymorphism in the human 5-HT, receptor 18. The method of claim 1, wherein the region of interest gene. Psychiatr Genet, 9(2), 105-106. is in the brain. 0289 Zhang, L. et al. (1999). Sex differences in expres 19. The method of claim 18, wherein the region of interest sion of serotonin receptors (Subtypes 1A and 2A) in rat is selected from the group consisting of the raphe nucleus, brain: a possible role of testosterone. Neuroscience, 94(1), dorsolateral prefrontal cortex, medial prefrontal cortex, 251-259. orbito-frontal cortex, anterior cingulate cortex, Subgenual 0290. Zhang, Y., et al. (2001) Segmentation of brain MR prefrontal cortex, temporal cortex, parietal cortex, occipital images through a hidden Markov random field model and cortex, amygdala, uncus, hippocampal formation, entorhinal the expectation—maximization algorithm. IEEE Trans cortex, parahippocampal gyrus, insula, dorsal raphe nuclei. Med Imaging 20: 45-57. and cerebellum. 0291 World Health Organization (2004) The global bur 20. (canceled) den of disease: 2004 update. 21. The method of claim 20, wherein the predetermined receptor binding potential threshold value is 39.9. 1. A method of determining whether a subject is afflicted 22. The method of claim 1, wherein the depressive with a depressive disorder comprising: disorder is major depression. (i) introducing into the Subject a positron emission tomog 23. The method of claim 1, wherein the subject is a human raphy (PET) radioligand capable of binding with a Subject. serotonin 5-HT, receptor, 24. The method of claim 23, wherein the human subject (ii) performing one or more PET scans of the subject; is a male Subject. (iii) determining, by analysis of the one or more PET 25. The method of claim 24, wherein the male subject had images, a receptor binding potential of the PET radio never undergone antidepressant treatment; or the male Sub ligand for the serotonin 5-HT, receptor in a region of ject had gone without antidepressant treatment for at least interest in the subject; four years. (iv) comparing the receptor binding potential value of the 26. (canceled) PET radioligand in the region of interest in the subject 27. A method of preparing a report classifying a Subject as to a predetermined receptor binding potential threshold having a depressive disorder or as not having a depressive value; and disorder which comprises: (V) classifying the Subject as having the depressive dis (i) receiving the data of one or more PET scans of the order or as not having the depressive disorder based on subject performed by a PET imaging device after a PET the comparison of step (iv), thereby determining radioligand for a serotonin 5-HT, receptor was intro whether the subject is afflicted with the depressive duced into the subject; disorder. (ii) processing the data to determine a receptor binding 2. The method of claim 1, wherein the PET radioligand is potential of the PET radioligand for the serotonin introduced by injection into the bloodstream of the subject. 5-HT, receptor in a region of interest in the subject 3. The method of claim 1, wherein the analysis of the one and comparing the receptor binding potential value to or more PET images in step (iii) is a computer analysis. a predetermined receptor binding potential threshold 4. The method of claim 1, wherein step (ii) further value; and comprises carrying out one or more MRI scans of the (iii) populating a report classifying the Subject. Subject. 28. A method of treating a subject afflicted with a depres 5. The method of claim 4, wherein the MRI images are sive disorder, comprising analyzed to define the boundaries of the region of interest. (a) determining whether the subject is afflicted with the 6. The method of claim 1, wherein the subject is classified depressive disorder comprising: as having the depressive disorder when the receptor binding (i) introducing into the Subject a positron emission potential value of the PET radioligand in the region of tomography (PET) radioligand capable of binding interest in the Subject is greater than the predetermined with a serotonin 5-HT, receptor; diagnostic threshold value. (ii) performing one or more PET scans of the subject; 7. The method of claim 1, wherein the subject is classified (iii) determining, by analysis of the one or more PET as not having the depressive disorder when the receptor images, a receptor binding potential of the PET binding potential value of the PET radioligand in the region radioligand for the serotonin 5-HT, receptor in a of interest in the subject is about the same or less than the region of interest in the Subject; predetermined diagnostic threshold value. (iv) comparing the receptor binding potential value of 8. The method of claim 1, wherein the PET radioligand the radioligand in the region of interest in the Subject contains a radioisotope selected from the group consisting of to a predetermined receptor binding potential thresh H, IC, 13N, 18F 123, 125I, 99mTc, 95Tc, Ill In, 62Cu, °Cu, old value; and Sc 7Ga, and Ga. (v) classifying the subject as afflicted with the depres 9. The method of claim 8, wherein the PET radioligand sive disorder when the receptor binding potential contains an ''C radioisotope or an F radioisotope. value of the radioligand in the Subject is greater than 10. (canceled) the predetermined diagnostic threshold value; and US 2017/007 1522 A1 Mar. 16, 2017 15

(b) treating the subject based on the determination obtained in step (a). 29.-31. (canceled) 32. The method of claim 28, wherein the subject is treated with an anti-depressant or psychotherapy. 33-35. (canceled)