<p>Aromatase is an enzyme that facilitates the conversion of androgens to estrogens and may play a role in mood and mental status. The main theme of this thesis is the imaging of brain aromatase by use of the PET technique. The PET tracer for aromatase, ¹¹C-labeled vorozole (VOZ) was developed and evaluated by with <i>in vitro</i> and <i>in vivo</i> methods. <i>In vitro</i> experiments using rat brain showed that VOZ was distributed in the medial amygdala, bed nucleus of the stria terminalis and medial preoptic area, regions of the brain known to be rich in aromatase and the K_D value was determined to be 0.60 nM. The <i>in vivo</i> PET study in rhesus monkey brain revealed that VOZ penetrated the blood-brain barrier and accumulated in the amygdala and hypothalamus. Taken together, VOZ is a good PET tracer for <i>in vivo</i> aromatase imaging with high affinity and high sensitivity.</p><p>This technique was applied to an investigation of brain aromatase under the physiological conditions simulating anabolic-androgenic steroid abuse. A significant increase in VOZ binding by anabolic-androgenic steroids was observed in the bed nucleus of stria terminalis and medial preoptic area in the rat brain. In contrast, no significant change in binding was observed in the medial amygdala. These results indicate that the manner of regulation of aromatase expression might be different in the bed nucleus of stria terminalis and medial preoptic area compared with that in the medial amygdala. The aromatase expression was suggested to be regulated through androgen receptors, as indicated in a study with flutamide treatment. The increased aromatase expression was seen in neurons. The PET study with anabolic steroid-treated rhesus monkeys also showed increased VOZ binding in the hypothalamus but not in the amygdala. The alteration of density of aromatase binding in the hypothalamic area could explain some psychological features of anabolic-androgenic steroid abusers.</p><p>Novel PET tracers for aromatase were developed and examined. The two newly synthesized ¹⁸F-labeled vorozole analogs, [¹⁸F]FVOZ and [¹⁸F]FVOO, displayed different characteristics. Both tracers showed similar binding pattern as VOZ; however, [¹⁸F]FVOO was metabolized very quickly, meaning that this tracer is not suitable as a PET tracer. On the other hand, [¹⁸F]FVOZ can be an appropriate PET tracer.</p><p>The role of aromatase in the human brain has not been clarified yet. To approach this problem by<i> in vivo</i> methods, we have just started PET studies to explore aromatase expression in humans.</p>

Aromatase is an enzyme that facilitates the conversion of androgens to estrogens and may play a role in mood and mental status. The main theme of this thesis is the imaging of brain aromatase by use of the PET technique. The PET tracer for aromatase, 11C-labeled vorozole (VOZ) was developed and evaluated by with in vitro and in vivo methods. In vitro experiments using rat brain showed that VOZ was distributed in the medial amygdala, bed nucleus of the stria terminalis and medial preoptic area, regions of the brain known to be rich in aromatase and the KD value was determined to be 0.60 nM. The in vivo PET study in rhesus monkey brain revealed that VOZ penetrated the blood-brain barrier and accumulated in the amygdala and hypothalamus. Taken together, VOZ is a good PET tracer for in vivo aromatase imaging with high affinity and high sensitivity. This technique was applied to an investigation of brain aromatase under the physiological conditions simulating anabolic-androgenic steroid abuse. A significant increase in VOZ binding by anabolic-androgenic steroids was observed in the bed nucleus of stria terminalis and medial preoptic area in the rat brain. In contrast, no significant change in binding was observed in the medial amygdala. These results indicate that the manner of regulation of aromatase expression might be different in the bed nucleus of stria terminalis and medial preoptic area compared with that in the medial amygdala. The aromatase expression was suggested to be regulated through androgen receptors, as indicated in a study with flutamide treatment. The increased aromatase expression was seen in neurons. The PET study with anabolic steroid-treated rhesus monkeys also showed increased VOZ binding in the hypothalamus but not in the amygdala. The alteration of density of aromatase binding in the hypothalamic area could explain some psychological features of anabolic-androgenic steroid abusers. Novel PET tracers for aromatase were developed and examined. The two newly synthesized 18F-labeled vorozole analogs, [18F]FVOZ and [18F]FVOO, displayed different characteristics. Both tracers showed similar binding pattern as VOZ; however, [18F]FVOO was metabolized very quickly, meaning that this tracer is not suitable as a PET tracer. On the other hand, [18F]FVOZ can be an appropriate PET tracer. The role of aromatase in the human brain has not been clarified yet. To approach this problem by in vivo methods, we have just started PET studies to explore aromatase expression in humans.

<p>An <i>in vitro</i> method was developed for measuring aromatase, based on binding of competitive aromatase inhibitor [¹¹C]vorozole to the active site of the enzyme. [¹¹C]Vorozole displayed high, specific binding <i>in vitro</i> to human placenta and human granulosa cells (GC), both fresh and frozen/thawed cells, provided correct procedures were used. High, specific binding was also observed in pig and rat ovaries, whereas binding in other tissues was unspecific and usually low. Aromatase concentrations measured by [¹¹C]vorozole binding correlated well to aromatase activity measured by [³H]water release from 1β[³H]androstenedione. </p><p>In human GC <i>in vitro</i>, low concentrations of 5α-dihydrotestosterone (DHT), but not of other androgens, stimulated aromatase activity measured by [³H]water release but had no effects on aromatase concentration measured by [¹¹C]vorozole binding. DHT may interact with aromatase differently than other androgens, perhaps by changing aromatase affinity to precursor. </p><p>In the rat estrous cycle, aromatase activity in ovarian homogenate, measured by [³H]water release, together with serum androstenedione and estradiol-17β, peaked between 6 and 13 h after onset of the light period of proestrus, the former activity being independent of radioactive substrate concentration. [¹¹C]Vorozole binding characteristics changed more rapidly than <i>de novo</i> synthesis of the enzyme. [¹¹C]Vorozole binding K_dshowed close inverse correlation to aromatase activity in ovarian homogenate and to serum estradiol-17β. Rapid changes in substrate affinity rather than changes in substrate concentration or <i>de novo</i> synthesis of the enzyme may thus be important for regulation of ovarian aromatase. </p><p>The [¹¹C]vorozole <i>in vivo</i> technique yields additional information compared with traditional in vitro techniques. </p>

An in vitro method was developed for measuring aromatase, based on binding of competitive aromatase inhibitor [11C]vorozole to the active site of the enzyme. [11C]Vorozole displayed high, specific binding in vitro to human placenta and human granulosa cells (GC), both fresh and frozen/thawed cells, provided correct procedures were used. High, specific binding was also observed in pig and rat ovaries, whereas binding in other tissues was unspecific and usually low. Aromatase concentrations measured by [11C]vorozole binding correlated well to aromatase activity measured by [3H]water release from 1β[3H]androstenedione. In human GC in vitro, low concentrations of 5α-dihydrotestosterone (DHT), but not of other androgens, stimulated aromatase activity measured by [3H]water release but had no effects on aromatase concentration measured by [11C]vorozole binding. DHT may interact with aromatase differently than other androgens, perhaps by changing aromatase affinity to precursor. In the rat estrous cycle, aromatase activity in ovarian homogenate, measured by [3H]water release, together with serum androstenedione and estradiol-17β, peaked between 6 and 13 h after onset of the light period of proestrus, the former activity being independent of radioactive substrate concentration. [11C]Vorozole binding characteristics changed more rapidly than de novo synthesis of the enzyme. [11C]Vorozole binding Kd showed close inverse correlation to aromatase activity in ovarian homogenate and to serum estradiol-17β. Rapid changes in substrate affinity rather than changes in substrate concentration or de novo synthesis of the enzyme may thus be important for regulation of ovarian aromatase. The [11C]vorozole in vivo technique yields additional information compared with traditional in vitro techniques.