Kelly A. Allers

Multifunctional Pharmacology of Flibanserin: Possible Mechanism of Therapeutic Action in Hypoactive Sexual Desire Disorder *

INTRODUCTION Flibanserin is a novel pharmacologic agent in late-stage clinical testing for hypoactive sexual desire disorder (HSDD) in premenopausal women. AIM The aim of this article is to review the hypothetical mechanism of action of flibanserin in HSDD. METHODS A literature review was conducted of all published works on flibanserin and on related studies of serotonin (5-HT)(1A) receptors and 5-HT(2A) receptors, including their actions on monoamines and on sexual function. MAIN OUTCOME MEASURES The main outcome measures are preclinical pharmacologic actions, especially changes in regional monoamines following treatment with flibanserin. RESULTS At clinically relevant doses, flibanserin acts predominantly at 5-HT(1A) receptors as an agonist and secondarily at 5-HT(2A) receptors as an antagonist. Additional binding actions within an order of magnitude of its 5-HT(1A) affinity, which are not likely to be clinically relevant, include weaker antagonist actions at 5-HT(2C) and 5-HT(2B) receptors, and less defined activity at dopamine (DA) D4 receptors. The 5-HT(1A) actions of flibanserin are only seen postsynaptically, which is unlike other agents such as buspirone that act at presynaptic 5-HT(1A) receptors. Furthermore, the postsynaptic actions of chronic flibanserin administration appear to demonstrate a preference for some populations of postsynaptic 5-HT receptors, particularly those that are located on the prefrontal cortex (PFC) pyramidal neurons, which regulate monoamine release in certain selective brain regions. CONCLUSIONS The regional selectivity of flibanserin results in a unique pattern of monoamine modulation. Sustained increases in baseline of DA and norepinephrine (NE) are observed in the PFC, and flibanserin dosing increases DA and NE levels above the basal changes. Conversely, flibanserin induces transient decreases in 5-HT levels in some brain areas such as the PFC, nucleus accumbens, and hypothalamus, but not in other brain areas such as the hippocampus. Therefore, since DA and NE are excitatory and 5-HT is inhibitory to sexual desire and arousal, it is tempting to postulate that the actions of flibanserin on serotonin receptors at the PFC pyramidal neurons, resulting in increased DA and NE yet reduced 5-HT in the PFC, are the mechanistic underpinnings of enhancing sexual desire in HSDD.

ORIGINAL RESEARCH—BASIC SCIENCE: Acute and Repeated Flibanserin Administration in Female Rats Modulates Monoamines Differentially Across Brain Areas: A Microdialysis Study

INTRODUCTION Hypoactive sexual desire disorder (HSDD) is defined as persistent lack of sexual fantasies or desire marked by distress. With a prevalence of 10% it is the most common form of female sexual dysfunction. Recently, the serotonin-1A (5-HT(1A)) receptor agonist and the serotonin-2A (5-HT(2A)) receptor antagonist flibanserin were shown to be safe and efficacious in premenopausal women suffering from HSDD in phase III clinical trials. AIM The current study aims to assess the effect of flibanserin on neurotransmitters serotonin (5-HT), norepinephrine (NE), dopamine (DA), glutamate, and gamma-aminobutyric acid (GABA) in brain areas associated with sexual behavior. METHODS Flibanserin was administered to female Wistar rats (280-350 g). Microdialysis probes were stereotactically inserted into the mPFC, NAC, or MPOA, under isoflurane anesthesia. The extracellular levels of neurotransmitters were assessed in freely moving animals, 24 hours after the surgery. MAIN OUTCOME MEASURES Dialysate levels of DA, NE, and serotonin from medial prefrontal cortex (mPFC), nucleus accumbens (NAC), and hypothalamic medial preoptic area (MPOA) from female rats. RESULTS Acute flibanserin administration decreased 5-HT and increased NE levels in all tested areas. DA was increased in mPFC and MPOA, but not in the NAC. Basal levels of NE in mPFC and NAC and of DA in mPFC were increased upon repeated flibanserin administration, when compared to vehicle-treated animals. The basal levels of 5-HT were not altered by repeated flibanserin administration, but basal DA and NE levels were increased in the mPFC. Glutamate and GABA levels remained unchanged following either repeated or acute flibanserin treatment. CONCLUSIONS Systemic administration of flibanserin to female rats differentially affects the monoamine systems of the brain. This may be the mechanistic underpinning of flibanserins therapeutic efficacy in HSDD, as sexual behavior is controlled by an intricate interplay between stimulatory (catecholaminergic) and inhibitory (serotonergic) systems.

Regional differences in neurovascular coupling in rat brain as determined by fMRI and electrophysiology

Increases in neuronal activity induce local increases in cerebral perfusion. However, our understanding of the processes underlying this neurovascular coupling remains incomplete and, particularly, how these vary across the brain. Recent work supports an important role for astrocytes in neurovascular coupling, in large part via activation of their metabotropic glutamate receptors (mGluR). Here, using a combination of functional magnetic resonance imaging (fMRI) and electrophysiology we demonstrate regional heterogeneity in the mechanisms underlying neurovascular coupling. Direct electrical stimulation of the rat hindpaw sensorimotor cortex induces blood oxygenation level dependent (BOLD) and cerebral blood volume (CBV) fMRI responses in several anatomically distinct cortical and subcortical structures. Following intraperitoneal administration of the type 5 mGluR antagonist, MPEP, both BOLD and CBV responses to cortical stimulation were significantly reduced, whilst the local field potential (LFP) responses remained largely constant. Spatially, the degree of reduction in fMRI responses varied between cortical and subcortical regions (primary cortex approximately 18% vs. striatum approximately 66%), and also between primary and secondary cortical areas ( approximately 18% vs. approximately 55%). Similarly, greater decreases in response amplitude were seen in the contralateral secondary cortex ( approximately 91%) and ipsilateral striatum (approximately 70%), compared to the primary cortex (approximately 44%). Following MPEP, a negative component of the BOLD and CBV responses became more apparent, suggesting that different mechanisms mediate vasodilatory and vasoconstrictory responses. Interestingly, under baseline conditions the quantitative relationship between fMRI and LFP responses in cortical and subcortical regions was markedly different. Our data indicate that coupling between neuronal and fMRI responses is neither empirically nor mechanistically consistent across the brain.

Neuroanatomical distribution of the melanocortin-4 receptors in male and female rodent brain

The melanocortin-4 receptor (MC4-R) plays a critical role in several physiological functions, from food intake, energy homeostasis, neuroendocrine and cardiovascular function, to sexual responses. The brain regions and the central neuronal pathways mediating the different actions of MC4-R remain largely unknown. We aimed to use immunocytochemistry using a specific antibody against rat MC4-R, to establish the detailed neuroanatomical distribution of MC4-R in brain slices of male and estrous female rats. We demonstrated that MC4-R-positive neurons were widely distributed in several brain regions including the cortex, thalamus, hypothalamus, and brainstem. In both male and female brains, MC4-R-positive cells were especially abundant in the hypothalamus, including the paraventricular hypothalamic nucleus, lateral septal nucleus, arcuate nucleus, supraoptic nucleus, medial preoptic area and lateral hypothalamic area. A moderate number of MC4-R-positive neurons were found in the piriform cortex, bed nucleus of the stria terminalis, medial and basolateral nuclei of amygdala, periaqueductal gray, red nucleus and raphe nucleus. A dimorphic sexual difference in the number of MC4-R-positive neurons was observed in some brain regions. In the medial preoptic area and arcuate nucleus, MC4-R-positive neurons were significantly more abundant in female than in males, whereas in the lateral hypothalamus the opposite proportion was observed. This is the first time the neuroanatomical distribution, and sex differences, of brain MC4-R localisation have been described. The distribution of MC4-R is consistent with the proposed roles of MC4-R-positive neurons and provides further information about the circuitry controlling food intake, energy balance and sexual responses in both males and females.

Neuroanatomical Evidence for a Role of Central Melanocortin-4 Receptors and Oxytocin in the Efferent Control of the Rodent Clitoris and Vagina

INTRODUCTION The clitoris and the vagina are the main peripheral anatomical structures involved in physiological changes related to sexual arousal and orgasm. Their efferent control and, more particularly, the neurochemical phenotype of these descending neuronal pathways remain largely uncharacterized. AIM To examine if brain neurons involved in the efferent control of the clitoris and the vagina possess melanocortin-4 receptor (MC4-R) and/or contain oxytocin (OT). METHODS Neurons involved in the efferent control of the vagina and clitoris were identified following visualization of pseudorabies virus (PRV) retrograde tracing. PRV was injected into the vagina and clitoris in adult rats in estrous. On the fifth day postinjection, animals were humanely sacrificed, and brains were removed and sectioned, and processed for PRV visualization. The neurochemical phenotype of PRV-positive neurons was identified using double or triple immunocytochemical labeling against PRV, MC4-R, and OT. Double and triple labeling were quantified using confocal laser scanning microscopy. MAIN OUTCOME MEASURE Neuroanatomical brain distribution, number and percentage of double-labeled PRV/MC4-R and PRV-/OT-positive neurons, and triple PRV-/MC4-R-/OT-labeled neurons. RESULTS The majority of PRV immunopositive neurons which also expressed immunoreactivity for MC4-R were located in the paraventricular and arcuate nuclei of the hypothalamus. The majority of PRV positive neurons which were immunoreactive (IR) for OT were located in the paraventricular nucleus (PVN), medial preoptic area (MPOA), and lateral hypothalamus. PRV positive neurons were more likely to be IR for MC4-R than for OT. Scattered triple-labeled PRV/MC4-R/OT neurons were detected in the MPOA and the PVN. CONCLUSION These data strongly suggest that MC4-R and, to a less extent, OT are involved in the efferent neuronal control of the clitoris and vagina, and consequently facilitate our understanding of how the melanocortinergic pathway regulates female sexual function.

Flibanserin Treatment Increases Appetitive Sexual Motivation in the Female Rat

INTRODUCTION Flibanserin is a mixed 5-HT1A agonist/5-HT2A antagonist that has been developed for the treatment of hypoactive sexual desire disorder in women. AIM To assess the acute and chronic dose-response effects of flibanserin on measures of sexual desire and copulation in ovariectomized rats primed with estradiol benzoate (EB) alone or in combination with progesterone (P). METHODS In Experiment 1, sexually experienced ovariectomized (OVX) rats at one testing site were rendered fully sexually receptive with EB + P priming and tested weekly with a sexually active male in bi-level pacing chambers following daily flibanserin treatment for 28 days. In Experiment 2, sexually experienced OVX rats at a different testing site received EB alone and were tested weekly with sexually active males following daily flibanserin treatment. MAIN OUTCOME MEASURES Female appetitive behaviors (solicitations, hops and darts, anogenital investigations), defensive behaviors, pacing, lordosis, and male copulatory responses (intromissions and ejaculations) were measured during each 30-minute copulation test. RESULTS Acute flibanserin or 1 week of chronic flibanserin treatment did not modify sexual responses in fully (EB + P) or partially (EB-alone) primed females. After 2 weeks of chronic treatment, fully primed females displayed significantly more solicitations than the three other groups. After 3 weeks of chronic treatment, a significant increase in female solicitations was observed in both hormone-treatment groups. CONCLUSION This study shows the first evidence that chronic, but not acute, flibanserin treatment augments appetitive sexual behaviors in OVX female rats primed with EB + P or EB alone. Given the positive effect of flibanserin in clinical trials, these results confirm previous reports that solicitations in the female rat are a predictive animal model of human female sexual desire.

Flibanserin and 8‐OH‐DPAT Implicate Serotonin in Association between Female Marmoset Monkey Sexual Behavior and Changes in Pair‐Bond Quality

INTRODUCTION Psychopathological origins of personally distressing, hypoactive sexual desire disorder (HSDD) in women are unknown, but are generally attributed to an inhibitory neural regulator, serotonin (5-HT). Flibanserin, a 5-HT(1A) agonist and 5-HT(2A) antagonist, shows promise as a treatment for HSDD. AIM To test the hypothesis that female marmoset sexual behavior is enhanced by flibanserin and diminished by 8-OH-DPAT, in order to evaluate the efficacy of serotonergic modulation of female sexual behavior in a pairmate social setting comparable to humans. METHODS Sexual and social behavior were examined in eight female marmoset monkeys receiving daily flibanserin (15 mg/kg), 8-OH-DPAT (0.1 mg/kg), or corresponding vehicle for 15-16 weeks in a counterbalanced, within-subject design, while housed in long-term, stable male-female pairs. MAIN OUTCOME MEASURES Marmoset pairmate interactions, including sexual and social behavior, were scored during weeks 5-6 of daily flibanserin, 8-OH-DPAT or vehicle treatment. 24-hour pharmacokinetic profiles of the drugs and their metabolites, as well as drug-induced acute symptoms of the 5-HT behavioral syndrome were also assessed. RESULTS Two-way analysis of variance reveals that flibanserin-treated females attract more male sexual interest (P=0.020) and trigger increased grooming (P=0.001) between partners. In contrast, 8-OH-DPAT-treated females show increased rejection of male sexual advances (P=0.024), a tendency for decreased male sexual interest (P=0.080), and increased aggression with their male pairmates (P=0.049). CONCLUSIONS While 8-OH-DPAT-treated female marmosets display decreased sexual receptivity and increased aggressive interactions with their male pairmates, flibanserin-treated female marmosets demonstrate increased affiliative behavior with their male pairmates. Such pro-affiliation attributes may underlie flibanserins effectiveness in treating HSDD in women.

Brain RegionSpecific Transcriptomic Markers of Serotonin1A Receptor Agonist Action Mediating Sexual Rejection and Aggression in Female Marmoset Monkeys

INTRODUCTION In a marmoset model of hypoactive female sexual function, we have shown that repeated administration of the serotonin (5-HT)-1A agonist R-(+)-8-hydroxy-2-(di-N-propylamino)tetralin (8-OH-DPAT) inhibits sexual receptivity in female marmoset monkeys and increases aggression toward the male pairmate. AIM The aims of this study are to investigate gene expression changes induced by 8-OH-DPAT in laser-microdissected brain areas that regulate female sexual function and to identify genes, functional gene classes, and pathways associated with 8-OH-DPAT-mediated inhibition of female sexual receptivity. METHODS Gene expression was measured in the medial prefrontal cortex (mPFC), medial preoptic area (mPOA), cornu ammonis-1 (CA1) area of the hippocampus (CA1), and dorsal raphé nucleus (DRN) of four 8-OH-DPAT-treated (0.1 mg/kg; daily administration for 16 weeks) and four vehicle-treated female marmosets using a marmoset-specific microarray (European Marmoset Microarray [EUMAMA]) and validated by real-time quantitative polymerase chain reaction (RTqPCR). Enriched functional gene classes were determined. In a parallel candidate gene approach, the expression of serotonergic candidate genes, i.e., the 5-HT1A, 5-HT2A, and 5-HT7 receptors and the 5-HT transporter (5-HTT), was measured by RTqPCR. MAIN OUTCOME MEASURES The main outcome is the differential expression of genes between 8-OH-DPAT- and vehicle-treated marmosets. RESULTS 8-OH-DPAT affected the gene classes important to neural development (mPFC, mPOA, and DRN), neurotransmission (mPOA), energy production (mPFC and mPOA), learning and memory (CA1), and intracellular signal transduction (DRN). Oxytocin (OXT) in the mPOA and 5-HTT in the DRN were strongly increased by 8-OH-DPAT. 5-HT1A tended to increase in the mPFC, while 5-HT7 was decreased in the CA1. CONCLUSIONS Brain region-specific alterations of gene expression regulating neural circuitries, energy demands, and learning processes are associated with 8-OH-DPAT-induced decrease in female sexual receptivity and increase in pairmate aggression. The role of OXT in the serotonergic regulation of female sexual behavior and partner interactions warrants attention in future studies.

Positron emission tomography assessment of 8-OH-DPAT-mediated changes in an index of cerebral glucose metabolism in female marmosets

As part of a larger experiment investigating serotonergic regulation of female marmoset sexual behavior, this study was designed to (1) advance methods for PET imaging of common marmoset monkey brain, (2) measure normalized FDG uptake as an index of local cerebral metabolic rates for glucose, and (3) study changes induced in this index of cerebral glucose metabolism by chronic treatment of female marmosets with a serotonin 1A receptor (5-HT(1A)) agonist. We hypothesized that chronic treatment with the 5-HT(1A) agonist 8-OH-DPAT would alter the glucose metabolism index in dorsal raphe (DR), medial prefrontal cortex (mPFC), medial preoptic area of hypothalamus (mPOA), ventromedial nucleus of hypothalamus (VMH), and field CA1 of hippocampus. Eight adult ovariectomized female common marmosets (Callithrix jacchus) were studied with and without estradiol replacement. In a crossover design, each subject was treated daily with 8-OH-DPAT (0.1mg/kg SC daily) or saline. After 42-49 days of treatment, the glucose metabolism radiotracer FDG was administered to each female immediately prior to 30 min of interaction with her male pairmate, after which the subject was anesthetized and imaged by PET. Whole brain normalized PET images were analyzed with anatomically defined regions of interest (ROI). Whole brain voxelwise mapping was also used to explore treatment effects and correlations between alterations in the glucose metabolism index and pairmate interactions. The rank order of normalized FDG uptake was VMH/mPOA>DR>mPFC/CA1 in both conditions. 8-OH-DPAT did not induce alterations in the glucose metabolism index in ROIs. Voxelwise mapping showed a significant reduction in normalized FDG uptake in response to 8-OH-DPAT in a cluster in medial occipital cortex as well as a significant correlation between increased rejection of mount attempts and reduced normalized FDG uptake in an overlapping cluster. In conclusion, PET imaging has been used to measure FDG uptake relative to whole brain in marmoset monkeys. Voxelwise mapping shows that 8-OH-DPAT reduces this index of glucose metabolism in medial occipital cortex, consistent with alterations in female sexual behavior.

Chronic systemic administration of serotonergic ligands flibanserin and 8-OH-DPAT enhance HPA axis responses to restraint in female marmosets

BACKGROUND Flibanserin, a novel serotonin (5-HT)(1A) agonist and 5-HT(2A) antagonist, has been shown to increase sexual desire and reduce distress in women with Hypoactive Sexual Desire Disorder (HSDD). In marmoset monkeys, flibanserin has demonstrated pro-social effects on male-female pairmates, while the classic 5-HT(1A) agonist 8-OH-DPAT suppresses female sexual behavior and increases aggressive interactions between pairmates. Activation of 5-HT(1A) and 5-HT(2A) receptors is known to stimulate the hypothalamic-pituitary-adrenal (HPA) axis. This study aims to characterize the effects of repeated flibanserin and 8-OH-DPAT administration on the marmoset HPA axis and to elucidate endocrine correlates of altered marmoset pair behavior. METHODS Adrenocorticotropic hormone (ACTH) and cortisol were examined at baseline and during 5-HT(1A) agonist and restraint challenges in 8 female marmoset monkeys receiving daily flibanserin (15mg/kg) and an additional 8 female marmosets receiving 8-OH-DPAT (0.1mg/kg) for 15-16weeks. Corresponding vehicle treatments were administered in a counterbalanced, within-subject design. All females were housed in stable male-female pairs. Treatment-induced changes in ACTH and cortisol levels were correlated with previously assessed marmoset pair behavior. RESULTS While morning basal cortisol levels and HPA responses to a 5-HT(1A) agonist challenge were not altered by chronic flibanserin or 8-OH-DPAT, both treatments increased the responsiveness of the marmoset HPA axis to restraint. Enhanced ACTH responses to restraint correlated with reduced sexual receptivity and increased aggression in 8-OH-DPAT-, but not in flibanserin-treated female marmosets. CONCLUSIONS Unaltered HPA responses to a 5-HT(1A) agonist challenge after chronic flibanserin and 8-OH-DPAT treatments indicate little or no de-sensitization of the HPA axis to repeated 5-HT(1A) manipulation. Chronic 8-OH-DPAT, but not flibanserin, leads to aggravated ACTH responses to stress that may contribute to anti-sexual and anti-social behavior between 8-OH-DPAT-treated females and their male pairmates. Despite similar flibanserin and 8-OH-DPAT induced ACTH responses to restraint stress, flibanserin-treated females show unchanged cortisol profiles. This is possibly due to flibanserins regional selectivity in 5-HT(1A) activation and concurrent 5-HT(2A) inhibition. The contrasting restraint-related cortisol responses emulate contrasting behavioral phenotypes of diminished pair-bond of 8-OH-DPAT-treated females compared to the more affiliative pair-bond of flibanserin-treated females.