A proof-of-concept randomized crossover clinical trial of a first-in-class vasopressin 1a receptor antagonist for PTSD: Design, methods, and recruitment.

Background: Almost eight million Americans suffer from Posttraumatic Stress Disorder (PTSD). Current PTSD drug therapies rely on repurposed antidepressants and anxiolytics, which produce undesirable side effects and have recognized compliance issues. Vasopressin represents a promising and novel target for pharmacological intervention. Logistical issues implementing a clinical trial for a novel PTSD pharmaceutical are relatively uncharted territory as trials concerning a new agent have not been published in the past several decades. All published trials have repurposed FDA-approved psychoactive medications with known risk profiles. Our recruitment challenges are discussed in this context. Methods: An 18-week proof-of-concept randomized crossover clinical trial of a first-in-class vasopressin 1a receptor antagonist (SRX246) for PTSD was conducted. All participants received SRX246 for 8 weeks, the placebo for 8 weeks, and the drug vs. placebo arms were compared. Participants were assessed every 2 weeks for PTSD symptoms as well as other medication effects. Results were expected to provide an initial demonstration of safety and tolerability in this clinical population and potentially clinical efficacy in SRX246-treated patients measured by Clinician Administered PTSD Scale (CAPS) score changes, clinical impression, and other indices compared to placebo. The primary hypothesis was that SRX246 would result in a clinically meaningful 10-point reduction in mean CAPS score compared to placebo. Discussion: This study is the first to investigate an oral vasopressin 1a receptor antagonist for PTSD. As a wave of PTSD clinical trials with new pharmaceutical compounds are beginning now, lessons learned from our recruitment challenges may be invaluable to these endeavors.

Safety and Tolerability of SRX246, a Vasopressin 1a Antagonist, in Irritable Huntington's Disease Patients-A Randomized Phase 2 Clinical Trial.

SRX246 is a vasopressin (AVP) 1a receptor antagonist that crosses the blood-brain barrier. It reduced impulsive aggression, fear, depression and anxiety in animal models, blocked the actions of intranasal AVP on aggression/fear circuits in an experimental medicine fMRI study and demonstrated excellent safety in Phase 1 multiple-ascending dose clinical trials. The present study was a 3-arm, multicenter, randomized, placebo-controlled, double-blind, 12-week, dose escalation study of SRX246 in early symptomatic Huntington's disease (HD) patients with irritability. Our goal was to determine whether SRX246 was safe and well tolerated in these HD patients given its potential use for the treatment of problematic neuropsychiatric symptoms. Participants were randomized to receive placebo or to escalate to 120 mg twice daily or 160 mg twice daily doses of SRX246. Assessments included standard safety tests, the Unified Huntington's Disease Rating Scale (UHDRS), and exploratory measures of problem behaviors. The groups had comparable demographics, features of HD and baseline irritability. Eighty-two out of 106 subjects randomized completed the trial on their assigned dose of drug. One-sided exact-method confidence interval tests were used to reject the null hypothesis of inferior tolerability or safety for each dose group vs. placebo. Apathy and suicidality were not affected by SRX246. Most adverse events in the active arms were considered unlikely to be related to SRX246. The compound was safe and well tolerated in HD patients and can be moved forward as a candidate to treat irritability and aggression.

Evidence of early vasogenic edema following minor head impact that can be reduced with a vasopressin V1a receptor antagonist.

BACKGROUND: Does minor head impact without signs of structural brain damage cause short-term changes in vasogenic edema as measured by an increase apparent diffusion coefficient (ADC) using diffusion weighted imaging? If so, could the increase in vasogenic edema be treated with a vasopressin V1a receptor antagonist? We hypothesized that SRX251, a highly selective V1a antagonist, would reduce vasogenic edema in response to a single minor head impact. METHODS: Lightly anesthetized male rats were subjected to a sham procedure or a single hit to the forehead using a closed skull, momentum exchange model. Animals recovered in five min and were injected with saline vehicle (n = 8) or SRX251 (n = 8) at 15 min post head impact and again 7-8 hrs later. At 2 h, 6 h, and 24 h post injury, rats were anesthetized and scanned for increases in ADC, a neurological measure of vasogenic edema. Sham rats (n = 6) were exposed to anesthesia and scanned at all time points but were not hit or treated. Images were registered to and analyzed using a 3D MRI rat atlas providing site-specific data on 150 different brain areas. These brain areas were parsed into 11 major brain regions. RESULTS: Untreated rats with brain injury showed a significant increase in global brain vasogenic edema as compared to sham and SRX251 treated rats. Edema peaked at 6 h in injured, untreated rats in three brain regions where changes in ADC were observed, but returned to sham levels by 24 h. There were regional variations in the time course of vasogenic edema and drug efficacy. Edema was significantly reduced in cerebellum and thalamus with SRX251 treatment while the basal ganglia did not show a response to treatment. CONCLUSION: A single minor impact to the forehead causes regional increases in vasogenic edema that peak at 6 h but return to baseline within a day in a subset of brain regions. Treatment with a selective V1a receptor antagonist can reduce much of the edema.

Facile and efficient preparation of organoimido derivatives of [Mo6O19]2- using accelerated reactions in Leidenfrost droplets.

Reaction acceleration is a hot topic in recent years since it is very useful for rapid reaction screening and small-scale synthesis on a short timescale. It is known that the rates of chemical reactions are often accelerated in confined volumes (small droplets or thin films) where the unique chemical reactivities of molecules at the air-droplet/thin film interface, usually different from that in the bulk and gas phases, play a dominant role in acceleration. The Leidenfrost effect was employed to create small levitated droplets with no net charge. These droplets can accelerate many kinds of organic reactions. Our first accelerated synthesis of a series of organoimido-functionalized polyoxometalate (POM) clusters using Leidenfrost droplets with product analysis by electrospray ionization mass spectrometry (ESI-MS) demonstrated that this method can be successfully extended to the synthesis of inorganic/organic hybrids, a very promising area for developing POM-based functional materials. Comparable amounts of synthetic products [Mo6O18(NC6H4R)]2- (R = H (6), m/z 477; p-i-C3H7 (7), m/z 498; p-OCH3 (8), m/z 492; p-NO2 (9), m/z 500) were prepared within minutes in Leidenfrost droplets versus in hours in the corresponding bulk reactions under the same reaction conditions in the presence of the DCC catalyst, suggesting that both concentration and interfacial effects are pivotal in causing reaction acceleration in the Leidenfrost droplet. Compared to the conventional bulk reactions, the acceleration factors (AFs) were 92, 136, 126, and 89 for the four model reactions (1)-(4), respectively. We also found out that substitution affects the rate of reactions occurring in droplets, and hence the magnitude of AF. The rates increase in the order of R = NO2 < H < i-C3H7 < OCH3, in which the electron-donating groups (i.e., R = OCH3, i-C3H7) on the benzene ring are more favorable to the reaction than the electron-withdrawing group (i.e., R = NO2). This experimental result is in good agreement with the DFT calculation which indicates that the free-energy barriers for the direct imidoylization of POM with RNH2 are linearly correlated with the basicity constants (pKb) of amines.

Treating head injury using a novel vasopressin 1a receptor antagonist.

Arginine vasopressin (AVP) is a chemical signal in the brain that influences cerebral vascular resistance and brain water permeability. Increases in AVP contribute to the pathophysiology of brain edema following traumatic brain injury (TBI). These effects are mediated through AVP V1a receptors that are expressed in cortical and subcortical brain areas. This exploratory study characterizes the effects of a novel, V1a receptor antagonist, AVN576, on behavioral and magnetic resonance imaging (MRI) measures after severe TBI. Male Sprague Dawley rats were impacted twice producing contusions in the forebrain, putative cerebral edema, and cognitive deficits. Rats were treated with AVN576 after initial impact for 5 days and then tested for changes in cognition. MRI was used to assess brain injury, enlargement of the ventricles, and resting state functional connectivity. Vehicle treated rats had significant deficits in learning and memory, enlarged ventricular volumes, and hypoconnectivity in hippocampal circuitry. AVN576 treatment eliminated the enlargement of the lateral ventricles and deficits in cognitive function while increasing connectivity in hippocampal circuitry. These data corroborate the extensive literature that drugs selectively targeting the AVP V1a receptor could be used to treat TBI in the clinic.

Nitrogen-Activated Oxidation in Nitrogen Direct Analysis in Real Time Mass Spectrometry (DART-MS) and Rapid Detection of Explosives Using Thermal Desorption DART-MS.

Direct analysis in real time mass spectrometry (DART-MS) was used to analyze an array of explosives including nitro-based explosives, peroxide explosives, and energetic heterocyclic compounds with different DART discharge gases (helium, argon, and nitrogen). Profound analyte oxidation was observed for particular compounds (TNT (9) and 2, 4-DNT (10)), whose mass spectra were completely dominated by the oxidation products when nitrogen was substituted for helium in DART analysis. This interesting phenomenon suggested that a highly oxidative environment provided by N2 DART ion source. A possible mechanism involved in nitrogen DART was proposed which may help further understanding the different chemistry involved in the ionization process. This work also presents a thermal desorption DART (TD-DART) configuration that can enable rapid, specific analysis of explosives from swipes. The screening of swipes with three different compositions (fiberglass, Hybond N+ membrane, and filter paper) showed that fiberglass swipe has the best performance which was then used for the subsequent TD-DART analysis. A direct comparison of TD-DART with traditional DART demonstrated that TD-DART indeed gives better response than traditional DART (provided that the distance between the DART source and mass spectrometer is the same) and will have wider applications than traditional DART.

[The clinical significance of typical reflux symptoms in diagnosing gastroesophageal reflux disease].

OBJECTIVE: To explore the clinical significance of typical reflux symptoms in the diagnosis of gastroesophageal reflux disease (GERD). METHODS: Consecutive patients older than 16 years, who initially visited department of gastroenterology at clinic of Peking University Third Hospital from May 9, 2012 to Dec 31, 2012, were required to complete a self-reported GERD questionnaire. Upper endoscopy was performed in some selected patients. RESULTS: A total of 18 987 patients were enrolled with a response rate of 91.5%. The prevalence of symptom-defined GERD was 13.6% (2 579/18 987). A total of 4 357 (22.9%) patients underwent the upper endoscopy, and the diagnostic rates of reflux esophagitis, Barrett's esophagus, peptic ulcer disease, and upper gastrointestinal malignancy were 13.1% (572/4 357), 1.8% (78/4 357), 10.5% (456/4 357), and 1.7% (75/4 357), respectively. The incidence of reflux esophagitis was 22.7% (216/951) in patients with reflux symptoms and 10.5% (356/3 406) (P < 0.001) in patients without reflux symptoms, 2.7% (26/951) and 1.5% (52/3 406), respectively (P = 0.013) for Barrett's esophagus; 6.8% (65/951) and 11.5% (391/3 406), respectively (P < 0.001) for peptic ulcer disease; 1.7% (16/951) and 1.7% (59/3 406), respectively (P = 0.917) for upper gastrointestinal malignancy. CONCLUSIONS: GERD is one of the major diseases at gastroenterology clinic. Typical reflux symptoms suggest a diagnosis of GERD. But some patients with peptic ulcer disease or upper gastrointestinal malignancy can also present typical reflux symptoms. Upper endoscopy is valuable to avoid the misdiagnosis of other disorders.

A novel V1a receptor antagonist blocks vasopressin-induced changes in the CNS response to emotional stimuli: an fMRI study.

BACKGROUND: We hypothesized that SRX246, a vasopressin V1a receptor antagonist, blocks the effect of intranasally administered vasopressin on brain processing of angry Ekman faces. An interaction of intranasal and oral drug was predicted in the amygdala. METHODS: Twenty-nine healthy male subjects received a baseline fMRI scan while they viewed angry faces and then were randomized to receive oral SRX246 (120 mg PO twice a day) or placebo. After an average of 7 days of treatment, they were given an acute dose of intranasal vasopressin (40 IU) or placebo and underwent a second scan. The primary outcome was BOLD activity in the amygdala in response to angry faces. Secondary analyses were focused on ROIs in a brain regions previously linked to vasopressin signaling. RESULTS: In subjects randomized to oral placebo-intranasal vasopressin, there was a significantly diminished amygdala BOLD response from the baseline to post-drug scan compared with oral placebo-intranasal placebo subjects. RM-ANOVA of the BOLD signal changes in the amygdala revealed a significant oral drug × intranasal drug × session interaction (F (1, 25) = 4.353, p < 0.05). Follow-up tests showed that antagonism of AVPR1a with SRX246 blocked the effect of intranasal vasopressin on the neural response to angry faces. Secondary analyses revealed that SRX246 treatment was associated with significantly attenuated BOLD responses to angry faces in the right temporoparietal junction, precuneus, anterior cingulate, and putamen. Exploratory analyses revealed that the interactive and main effects of intranasal vasopressin and SRX246 were not seen for happy or neutral faces, but were detected for aversive faces (fear + anger) and at a trend level for fear faces. CONCLUSION: We found confirmatory evidence that SRX246 has effects on the amygdala that counter the effects of intranasal vasopressin. These effects were strongest for angry faces, but may generalize to other emotions with an aversive quality.

Pharmacokinetics and metabolism of SRX246: a potent and selective vasopressin 1a antagonist.

SRX246 is a potent, highly selective, orally bioavailable vasopressin 1a receptor antagonist that represents a novel mechanism of action for the treatment of mood disorders. The compound previously showed efficacy in animal models of mood disorders and excellent safety and tolerability in healthy volunteers in phase I clinical trials. In this study, SRX246 was further characterized in rats and dogs. In vitro determinations of permeability, protein binding, hepatocyte metabolism, and cytochrome P450 enzyme inhibition and in vivo assessments of pharmacokinetics were conducted. In parallel artificial membrane permeability assay (PAMPA) and PAMPA-blood-brain barrier models, SRX246 was comparable to highly permeable, orally active pharmaceuticals. SRX246 hydrochloride salt was 95.5 ± 1.7%, 95.9 ± 1.3%, and 98.6 ± 0.4% bound to rat, dog, and human serum proteins, respectively, and was stable in serum after a 4 h incubation at 37°C. P450 enzyme inhibition results showed a very low potential for drug-drug interactions. Metabolism in primary hepatocytes demonstrated that SRX246 was stable in humans and moderately metabolized in dogs and rats. Plasma pharmacokinetics findings showed a half-life (T½ ) of 2 and 6 h in rat and dog, respectively. Rat brain levels following a single oral dose were approximately 20% of plasma values with a T½ of 6 h. The observed profile for SRX246 supports further development.

Synthesis and evaluation of potent and selective human V1a receptor antagonists as potential ligands for PET or SPECT imaging.

SRX246 is a potent, highly selective human vasopressin V1a antagonist that crosses the blood-brain barrier in rats. CNS penetration makes SRX246 an ideal candidate for potential radiolabeling and use in visualization and characterization of the role of the V1a receptor in multiple stress-related disorders. Before radiolabeling studies, cold reference analogs of SRX246 were prepared. This study describes the synthesis and in vitro screening for human V1a receptor binding and permeability of fluoro, iodo, and methyl reference compounds for SRX246 and the preparation of a tin precursor. For each compound, the potential utility of corresponding radiolabeled analogs for PET and SPECT imaging is discussed.

Genome-wide analysis of DHEA- and DHT-induced gene expression in mouse hypothalamus and hippocampus.

Dehydroepiandrosterone (DHEA) is the most abundant steroid in humans and a multi-functional neuroactive steroid that has been implicated in a variety of biological effects in both the periphery and central nervous system. Mechanistic studies of DHEA in the periphery have emphasized its role as a prohormone and those in the brain have focused on effects exerted at cell surface receptors. Recent results demonstrated that DHEA is intrinsically androgenic. It competes with DHT for binding to androgen receptor (AR), induces AR-regulated reporter gene expression in vitro, and exogenous DHEA administration regulates gene expression in peripheral androgen-dependent tissues and LnCAP prostate cancer cells, indicating genomic effects and adding a level of complexity to functional models. The absence of information about the effect of DHEA on gene expression in the CNS is a significant gap in light of continuing clinical interest in the compound as a hormone replacement therapy in older individuals, patients with adrenal insufficiency, and as a treatment that improves sense of well-being, increases libido, relieves depressive symptoms, and serves as a neuroprotective agent. In the present study, ovariectomized CF-1 female mice, an established model for assessing CNS effects of androgens, were treated with DHEA (1mg/day), dihydrotestosterone (DHT, a potent androgen used as a positive control; 0.1mg/day) or vehicle (negative control) for 7 days. The effects of DHEA on gene expression were assessed in two regions of the CNS that are enriched in AR, hypothalamus and hippocampus, using DNA microarray, real-time RT-PCR, and immunohistochemistry. RIA of serum samples assessed treatment effects on circulating levels of major steroids. In hypothalamus, DHEA and DHT significantly up-regulated the gene expression of hypocretin (Hcrt; also called orexin), pro-melanin-concentrating hormone (Pmch), and protein kinase C delta (Prkcd), and down-regulated the expression of deleted in bladder cancer chromosome region candidate 1 (Dbccr1) and chitinase 3-like 3 (Chi3l3). Two-step real-time RT-PCR confirmed changes in the expression of three genes (Pmch, Hcrt and Prkcd) using the same RNA sample employed in the microarray experiment. Immunohistochemistry showed augmentation of prepro-hypocretin (pHcrt) neuropeptide protein expression by DHEA and DHT in hypothalamus, consistent with the localization of orexin neurons. In hippocampus, DHT down-regulated the expression of Prkcd, while DHEA did not have significant effects. RIA results supported the view that DHEA-induced effects were mediated through AR. The current study identified neurogenomic effects of DHEA treatment on a subset of genes directly implicated in the regulation of appetite, energy utilization, alertness, apoptosis, and cell survival. These changes in gene expression in the CNS represent a constellation of effects that may help explain the diverse benefits attributed to replacement therapy with DHEA. The data also provide a new level of detail regarding the genomic mechanism of action of DHEA in the CNS and strongly support a central role for the androgen receptor in the production of these effects. More broadly, the results may be clinically significant because they provide new insights into processes that appear to mediate the diverse CNS effects attributed to DHEA.

Vasopressin antagonists as anxiolytics and antidepressants: recent developments.

A compelling case for the potential utility of vasopressin (AVP) antagonists as a novel therapeutic class for the treatment of stress-related affective illness has emerged based on observations in depressed individuals, findings in animal models of anxiety and depression, and an understanding of changes in hypothalamic-pituitary-adrenal (HPA) axis regulation under chronic stress. The scientific bases for vasopressin antagonists as a pharmacotherapy for anxiety and depression include: 1) the neuroadaptation and dysregulation of HPA function that accompanies chronic stress in affected humans and in animal models of anxiety and depression, 2) recognition that AVP, not corticotrophin releasing factor (CRF), drives HPA function associated with chronic psychological stress, 3) the CNS localization of vasopressin V1a and V1b receptors in limbic system regions involved in HPA regulation and control of social behaviors, and 4) preclinical data showing efficacy in animal models employed as screens for anxiolytic and antidepressant activity. The public health need for new pharmaceutical treatments for stress-related affective illness is well documented. In the United States alone, anxiety and depression affect some 40 million people each year and carry a conservatively estimated annual total economic burden of at least $125 billion. Existing pharmacotherapies for both indications are not uniformly effective and frequently have undesirable side effects. These limitations demonstrate that a new treatment approach through vasopressin receptor antagonism in the CNS may offer significant opportunities for improved outcomes. In this review, the development of compounds in this class since 2005 is considered. The most advanced clinical candidates and newer compounds described in recent patents are presented.

Azetidinones as vasopressin V1a antagonists.

The azetidinone LY307174 (1) was identified as a screening lead for the vasopressin V1a receptor (IC50 45 nM at the human V1a receptor) based on molecular similarity to ketoconazole (2), a known antagonist of the luteinizing hormone releasing hormone receptor. Structure-activity relationships for the series were explored to optimize receptor affinity and pharmacokinetic properties, resulting in compounds with Ki values <1nM and brain levels after oral dosing approximately 100-fold higher than receptor affinities.

Orally active vasopressin V1a receptor antagonist, SRX251, selectively blocks aggressive behavior.

Arginine vasopressin functions as a neurochemical signal in the brain to affect social behavior. There is an expanding literature from animal and human studies showing that vasopressin, through the vasopressin 1A receptor (V1A), can stimulate aggressive behavior. Using a novel monocylic beta lactam platform, a series of orally active vasopressin V1a antagonists was developed with high affinity for the human receptor. SRX251 was chosen from this series of V1a antagonists to screen for effects on serenic activity in a resident-intruder model of offensive aggression. Resident, male Syrian golden hamsters were given oral doses of SRX251 or intraperitoneal Manning compound, a selective V1a receptor antagonist with reduced brain penetrance, at doses of 0.2 microg, 20 microg, 2 mg/kg or vehicle. When tested 90-120 min later, SRX251, but not Manning compound, caused a significant dose-dependent reduction in offensive aggression toward intruders as measured by latency to bite and number of bites. The reduction in aggression persisted for over 6 h and was no longer present 12 h post treatment. SRX251 did not alter the amount of time the resident investigated the intruder, olfactory communication, general motor activity, or sexual motivation. These data corroborate previous studies showing a role for vasopressin neurotransmission in aggression and suggest that V1a receptor antagonists may be used to treat interpersonal violence co-occurring with such illness as ADHD, autism, bipolar disorder, and substance abuse.

Dehydroepiandrosterone and its metabolites: differential effects on androgen receptor trafficking and transcriptional activity.

Dehydroepiandrosterone (DHEA) is a multi-functional steroid that has been implicated in a broad range of biological effects in humans and rodents. Recent studies demonstrated that DHEA acts genomically through the androgen receptor (AR) in addition to its well-known effects on cell surface receptors. However, the relative contribution of DHEA and its major metabolites, including DHEA-Sulfate (DHEA-S), 7alpha-OH-DHEA, 7beta-OH-DHEA, 7-oxo-DHEA, androstenedione (Adione), and androstenediol (Adiol), in the production of genomic effects remains controversial, in part because the metabolism of DHEA varies in different cells and tissues. In the current study, the ability of DHEA and its metabolites to promote AR intracellular trafficking and regulate AR-mediated reporter gene expression, which are characteristic effects of androgens, was determined. Intracellular trafficking of AR-GFP protein was assessed in COS-7 cells while AR transcriptional activity was tested in CV-1 cells transiently co-transfected with AR expression plasmid and an MMTV-ARE-CAT reporter. The results demonstrated that DHEA, the 3beta-HSD metabolite Adione, and the 17beta-HSD metabolite Adiol, were androgenic. Each promoted AR-GFP intracellular trafficking, the formation of nuclear clusters, and AR-dependent transcriptional activity in a dose-dependent manner. In contrast, DHEA-S, 7alpha-OH-DHEA, 7beta-OH-DHEA, and 7-oxo-DHEA were ineffective and exhibited minimal androgenic activity, even at relatively high concentrations (10(-6) M). These results provide the first systematic comparison of the (i) androgenic activity of DHEA and its sulfated and hydroxylated metabolites, (ii) relative androgenicity of DHEA itself vs. the established androgens Adione and Adiol, and (iii) ability of DHEA and its major metabolites to promote AR-GFP intracellular trafficking. In addition to partitioning DHEA and its metabolites into compounds with (DHEA, Adione, Adiol) and without (DHEA-S, 7alpha-OH-DHEA, 7beta-OH-DHEA, and 7-oxo-DHEA) androgenic activity, the findings improve our understanding of the intracellular processes mediating the genomic effects of DHEA through AR.

ERbeta protein expression in female cynomolgus monkey and CF-1 mouse brain: Western analysis.

In humans and rodents, multiple ERbeta variants with sizes ranging from 477-549 amino acids (aa) have been described. The identification of these variants in target tissues has important implications for estrogen signaling and cellular responsiveness. Western blot analysis using two anti-ERbeta antibodies specific for mammalian ERbeta sequences (PA1-310B and PA1-311) was employed to examine ERbeta protein expression in neural tissues from ovariectomized (OVX) cynomolgus macaques and CF-1 mice as well as to assess potential regulatory effects of acute and extended estradiol (E(2)) treatment. In hypothalamic extracts from both species, a single ERbeta immunoreactive (ERbeta-ir) band was detected at approximately 54 kDa, corresponding to the expected molecular weight for ERbeta477 and/or 485. In cynomolgus females, oral E(2) administration for 16 weeks had no apparent effect on hypothalamic ERbeta protein expression. In mouse, a single injection of E(2) did not change hypothalamic ERbeta protein levels 1.5, 4, 8, 16, or 24 h after injection. Extending the hormonal treatment to 4 or 21 days in OVX female mice also had no effect on the level of hypothalamic ERbeta protein. Additional regional analyses in female mouse brain with PA1-310B antibody showed that a second, 59 kDa ERbeta-ir band was present in cortex, striatum, hippocampus, and amygdala that could represent one or both of the larger ERbeta variants (530 and 549aa). The expression level of the second ERbeta isoform exhibited regional variation, with the strongest immunoreactivity detected in cortex and amygdala. Elucidating the functions of these ERbeta isoforms in the CNS will facilitate our understanding of the tissue- and promoter-specific actions of estrogen.

DHEA and DHEA sulfate differentially regulate neural androgen receptor and its transcriptional activity.

The mechanism of action of dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEA-S), two interconvertable neurosteroids, has not been fully characterized in the central nervous system (CNS). Previous studies demonstrated that DHEA was intrinsically androgenic, suggesting that it may act through a genomic pathway. However, it is not known whether DHEA-S also produces androgenic effects, an important question given that the concentration of DHEA-S in brain is some 7-12 times that of DHEA. The current study compared the potential androgenic effects of DHEA-S with DHEA by examining their capacity to induce two characteristic effects of an androgenic compound. These included the ability to (1) up-regulate neural androgen receptor (AR) protein level in mouse brain and immortalized GT1-7 hypothalamic cells and (2) assess their effect on reporter gene expression through AR in CV-1 cells cotransfected with pSG5-AR and pMMTV-ARE-CAT reporter. Semi-quantitative Western blot analysis showed that DHEA treatment significantly augmented AR in mouse brain and GT1-7 cells in a dose-dependent manner and that these effects were not blocked by trilostane (TRIL), a known 3beta-hydroxysteroid dehydrogenase inhibitor. DHEA also promoted AR-mediated reporter gene expression as a function of dose and the effect was comparable with or without the addition of TRIL. In contrast, DHEA-S treatment failed to increase AR level in the mouse brain or GT1-7 cells and modestly induced AR-mediated reporter gene expression only at substantially elevated concentrations compared to DHEA. The findings demonstrate that DHEA is capable of exerting androgenic effects through AR while the androgenicity of DHEA-S is negligible. The implications of the results for models of the mechanism of action of DHEA and its sulfate ester, DHEA-S, in the brain are considered.

Dehydroepiandrosterone upregulates neural androgen receptor level and transcriptional activity.

The mechanism of action of dehydroepiandrosterone (DHEA), a neuroactive neurosteroid synthesized in the brains of humans and other mammals, has not been fully characterized in the adult brain. Although well known for modulatory effects on GABA(A), NMDA, and sigma(1) receptors, studies in both CNS and peripheral target cells suggest that DHEA also may exert genomic effects via the androgen receptor (AR). The current study tested the hypothesis that DHEA was capable of producing androgenic effects in the CNS by assaying its ability to induce three characteristic effects of an androgenic compound. These included the ability to upregulate neural AR protein level in mouse brain and immortalized GT1-7 hypothalamic cells, the capacity to induce transcriptional activity through AR in CV-1 cells transfected with an MMTV-ARE-CAT reporter, and competition for recombinant AR binding in a radioligand binding assay. The results showed that DHEA treatment significantly augmented AR both in vivo and in vitro, and that this effect was not blocked by trilostane (TRIL), a known 3beta-hydroxysteroid dehydrogenase (3beta-HSD) inhibitor. DHEA also promoted AR-mediated CAT reporter expression and competed with dihydrotestosterone (DHT) for binding to recombinant AR in a cell-free system. These data indicate that DHEA possesses intrinsic androgenic activity that is potentially independent of metabolic conversion to other androgens, and that it can affect gene function through the AR. In combination with its modulation of neurotransmitter receptors at the cell membrane level, the findings suggest that the mechanism of action of DHEA in the brain can involve a "crosstalk" cellular signaling system that involves both nongenomic and genomic components.