**4.1 Apomorphine**

82 Sexual Dysfunctions – Special Issue

patient will rate her subjective feelings of arousal quite low, but tends to show a normal genital response (Laan et al. 2008; Rellini et al. 2006). Classifying patients into subgroups of mainly genital arousal, or subjective arousal dysfunction in studies using VPA begins to show some differences, but subjective assessments remain the best diagnostic tools (Both et al. 2010; Salonia et al. 2010). In healthy women, there is not always agreement between subjective and physiologically measured responses either, perhaps due to interoceptive

Even though there is a wealth of information on healthy arousal responses, there is no evidence based hypothesis for the neuropathology of FSAD. Clear differences in sympathetic responsiveness has been demonstrated in patients as compared to controls using procedures such as hyperventilation or extreme exercise (Brotto et al. 2009; Meston 2000). This points to a pathology in the systemic autonomic nervous system, or central-

The majority of models above cannot provide translational disease models for FSAD, since vaginal vasocongestion is not affected in a large number of women with FSAD, and the likely pathology is within the circuitry of the autonomic or central nervous system. Only one technique above has demonstrated that it can actually distinguish between healthy controls

Slow oscillations in vaginal blood flow: In this model, laser Doppler flowmetry or vaginal plethysmograph can be used to record blood flow within the vaginal wall (Allers et al. 2010d; Allers et al. 2010c). Rather than assessing peak amplitudes, rate or other typical measures, the trace is analysed by fast Fourier transform (FFT) to elucidate the oscillatory charactistics of flow. This technique is well established in vascular research and these oscillations are known to reflect autonomic nervous system input to the tissue being studied. In this way, these measures are a surrogate of autonomic input. When measured in the vagina, the changes seen during sexual arousal paradigms are specific to the vagina, meaning they do not occur in other tissues simultaneously. This method has been used to differentiate human patients, correlates highly with subjective arousal in both healthy and FSAD women, and has been used in animals to show arousal inducing effects of apomorphine and melanotan II, in addition to natural arousal (exposure to sexually active male). Upon apomorphine administration animals in metestrus respond with increased slow oscillations in this model, at the same doses that induce restoration of sexual behavior in a partner-preference test (Hawcock et al. 2010). This model has not yet been used clinically to demonstrate restoration of natural arousal in FSAD women with a drug candidate, but has

and women with FSAD, and has been demonstrated to have predictive utility.

great potential as a translational model for investigation of drugs to treat FSAD.

The process of drug discovery requires that a target (eg receptor, enzyme, etc.) be identified with a reasonable hypothesis for why it is engaged in a given disease state. This hypothesis is then rigorously tested within the laboratory with tool compounds and drug candidates. Alternatively, and typical of the sexual health field, a prosexual side effect is noticed for a given drug candidate, and this is quickly followed up to assess its potential for therapeutic use in sexual medicine. This process leads to a substantial amount of research into pathways, disease states, and pharmacological mechanisms regarding a particular target being generated. Within sexual health groups in the pharmaceutical industry recent targets have included dopamine receptors, serotonin receptors, and melanocortin receptors. The

differences in attending to one's own genital physiology.

autonomic interface, rather than in the genitalia.

**4. Lessons from drug discovery** 

Drug development teams have adopted multiple strategies for assessing dopamine receptors as a drug target, including assessing the roles of individual dopamine receptor subtypes, dopamine reuptake, or using a classical dopamine receptor agonist (apomorphine) in different formulations. Apomorphine is a non-selective dopamine agonist was first discovered in 1869 and over time has been used as an emetic, a treatment for alcohol and morphine addictions, and to improve symptoms of Parkinson's Disease (Subramony 2006).

Dopamine has long been known to be a modulator of sexual function. There are decades of literature reporting pro-sexual effects of dopamine agonists but also reports of the same paradigms producing reduced sexual behavior. Much research has focussed on elucidating the hormone dependence of effects, the regions of the brain where increased or decreased dopamine occurs during sexual contact in efforts to investigate the real role of dopamine in sexual function and where discrepancies in data may come from. Many review articles have highlighted ongoing questions related to dopamine in sexual function (Meisel et al. 2006; Paredes et al. 2004; Peeters et al. 2008; Pfaus 2009a; Stolzenberg et al. 2011b; Stolzenberg et al. 2011a).

Regardless of the scientific debates over how dopamine regulates sexual function, clinical evidence has indicated that stimulating dopamine receptors may provide help for women with sexual dysfunction (Bechara et al. 2004; Caruso et al. 2004a). For this reason, studies were undertaken to investigate further why apomorphine can have opposing effects on sexual behavior in rodents.

Using a partner preference paradigm in which a rat chooses to actively investigate either a sexually vigorous male or a castrated male, a measure of active investigation can be utilised to determine sexual interest compared to social interest. In this paradigm, female rats that are ovariectomised and given a sub-hormone prime to resemble metestrus do not show a preference for either male rat. Upon apomorphine treatment, a dose dependent increase in preference for the sexually vigorous male is observed. When this study is run with animals that have been fully hormonally primed to resemble behavioral estrous, they show a clear preference for the sexually vigorous male, which is decreased by apomorphine treatment (Hawcock et al. 2010). These data indicate that when ALL other conditions are equal dopamine receptor agonism can have the exact opposite effect simply by artificially placing the animal into different stages of the estrous cycle.

Further investigations suggest that the opposing effects of apomorphine occur in the naturally cycling animal in metestrus compared to estrous. Using the FFT analysis of laser doppler flowmetry in rats, these differential effects of apomorphine are also present. Metestrus animals had a significant increase in slow oscillatory activity of blood flow, which in this model is indicative of sexual arousal (Allers et al. 2010d). Furthermore, this increase could not be blocked by the peripheral antagonist domperidone, but could be attenuated with the centrally acting antagonist haloperidol, indicating apomorphine's actions did originate in the brain (Allers et al. 2010c). In estrous animals, apomorphine elicited a decrease in slow oscillatory activity indicating decreased arousal. The dose ranges in both

Paradigms for Preclinical Investigations

oxytocin neurons.

**4.3 Flibanserin** 

the positive control, paroxetine (Kennedy 2010).

Goldfischer et al. 2009; Jolly et al. 2009).

of Female Sexual Function and Dysfunction (HSDD and FSAD) 85

most evidence currently points to action within either the medial pre-optic area (MPOA) or the paraventricular nuclues (PVN) or both. Behavioral studies in pacing chambers have demonstrated that peripheral administration of bremelanotide increases proceptive behaviors in female rats with different hormone primes. In addition, injection of the drug directly into the MPOA results in the same effect (Pfaus et al. 2007). In keeping with this data, peripheral injection of bremelanotide results in increased activation of MPOA neurons as measured by c-fos. In addition to bremelanotide's actions on proceptivity, the parent compound MTII has also been demonstrated to induce arousal in rats using the laser

In male rats, a similar study demonstrates that following administration of bremelanotide cfos activation occurs within the PVN of the hypothalamus (Molinoff et al. 2003). To assess if this is a potential pathway in females, further investigation was undertaken in naturally cycling estrus rats. Pseudorabies virus (PRV) injection to the clitoris and vagina resulted in transsynaptic labeling present in both the PVN and the MPOA. Furthermore, neurons that were double-labeled for PRV and the melanocortin 4 receptor were found in both the PVN and MPOA, indicating melanocortin pathways exist from both of these regions to the genitalia (Gelez et al. 2010). Between 4 and 8% of the PRV labeled neurons were triplelabeled for both the melanocortin 4 receptor and oxytocin. These data demonstrate direct pathways from both the PVN and the MPOA to the genitalia that could be part of bremelanotide's mechanism of action through activation of melanocortin 4 receptors on

Flibanserin was discovered in 1990 as part of a program investigating targets for depression (Borsini et al. 1997). This compound was developed based on a very sound rationale for why agonist activity at post-synaptic 5-HT1A receptors and antagonism of 5-HT2A receptors combined would be beneficial to patients with major depressive disorder (Borsini et al. 2002). Unfortuantely, during Phase II trials for depression, flibanserin was not superior to

Within the Phase II trials, patients were given the ASEX questionnaire to assess sexual function. Flibanserin treatment improved sexual function in 70% of the patients. Development of flibanserin for depression was discontinued but restarted development for the indication of hypoactive sexual desire disorder. Following several Phase III clinical trials with flibanserin in premenopausal women, the data indicating increased desire and decreased distress following chronic flibanserin treatment is substantial (Clayton et al. 2009;

Following the reassignment of this drug, the search for a mechanism of action began. Within sexual function research, 5-HT1A receptor agonism has long been known to reduce sexual behavior (Ahlenius et al. 1989; Mendelson et al. 1986; Uphouse et al. 1991). The finding that a 5-HT1A agonist is prosexual in women was puzzling. Treatment of rats in pacing chambers either at full estrous priming or sub-hormonal priming also indicated that rats increased proceptive behaviors with chronic flibanserin treatment, and hence, sexual motivation – eliminating the possibility of a species difference in 5-HT1A actions (Allers et al. 2010b; Greggain et al. 2010). Acute dosing of flibanserin has no effect on sexual behavior in rodents. In pair-bonded marmosets, a study was conducted to compare the effects of chronic flibanserin treatment with that of a commonly used 5-HT1A agonist, 8-OH-DPAT. In this study, flibanserin induced increased affiliative behavior in both pair mates, although only

doppler method with FFT analysis of slow oscillatory activity (Allers et al. 2010d).

metestrus and estrous animals which had effects in this model are the same as those in the partner-preference model.

An electrophysiological study was undertaken to further understand the underlying mechanisms for the observed differences in apomorphine actions. Neurons from the paraventricular nucleus, a nucleus important for sexual function and hormonally regulated, were studied in animals from all four stages of the estrous cycle. Firing rates from this nucleus varied substantially across the estrous cycle, with metestrous rates being the highest. In addition subpopulations of neurons were identified: slow neurons which increase in response to apomorphine, and fast neurons which decrease in response to the drug. A greater proportion of the fast neurons were evident in metestrous, accounting for the higher mean firing rate, and leading to relatively greater decreases upon apomorphine (Richards et al. 2010).

Taken together, these data suggest that during metestrous, a neurological 'stop' signal has been physiologically delivered to the animal which is reflected the animal's behavioral disinterest in a sexual partner. This 'stop' signal is sensitive to, and can be reversed by apomorphine administration. This reversal manifests as increased interest in a sexual partner and increased sexual arousal. An underlying mechanism may be the inhibition of a fast-firing population of neurons within the paraventricular nucleus of the brain.

#### **4.2 Bremelanotide**

The peptide -melanocyte stimulating hormone (MSH) is a product of the proopiomelanocortin pro-hormone. This peptide has long been known to be involved in regulation of energy homeostasis and has been suggested as a target for number medical indications (Hedlund 2004).

In the mid-1980s, a group at the University of Arizona synthesized two highly potent MSH analogues (Hadley et al. 1998). One compound, deemed Melanotan I (MTI) was licensed out and further characterised for utility as a tanning drug, given the known role of MSH in pigmentation. A further analogue, Melanotan II (MTII) was developed which was smaller and the hope was that this would aid in its absorption and tissue distribution. The investigator decided to assess for himself whether this second analogue had the tanning capability seen with MTI and proceeded to dose himself. While it is unclear whether he did achieve a tan, what the investigator reports was an "unrelenting" erection lasting 8 hours. Not long after, this compound was licensed out for further development as a sexual dysfunction treatment candidate. PT-141 is the active metabolite of MTII and ultimately became the drug development lead compound and was renamed bremelanotide.

Clincal trials in women have demonstrated that bremelanotide increases sexual desire and arousal in women with arousal disorders (Diamond et al. 2004; Diamond et al. 2006; Safarinejad 2008). In one study, using vaginal plethysmography to assess vasocongestion, even though subjective scores were increased over placebo, there was no change in vasocongestion measures as compared to controls, confirming that vaginal vasocongestion is not a suitable method for assessing efficacy of compounds (Diamond et al. 2006).

Prior to the discovery and development of bremelanotide, melanocortin receptors were not considered to be of great interest within the sexual medicine field. Since that time, a surge of interest has appeared, and along with it a boost in scientific research investigating the mechanisms involved.

Bremelanotide is an agonist at melanocortin 3 and 4 (MC3 and MC4) receptors, whose primary localisation is in the hypothalamic regions of the brain (Molinoff et al. 2003). The most evidence currently points to action within either the medial pre-optic area (MPOA) or the paraventricular nuclues (PVN) or both. Behavioral studies in pacing chambers have demonstrated that peripheral administration of bremelanotide increases proceptive behaviors in female rats with different hormone primes. In addition, injection of the drug directly into the MPOA results in the same effect (Pfaus et al. 2007). In keeping with this data, peripheral injection of bremelanotide results in increased activation of MPOA neurons as measured by c-fos. In addition to bremelanotide's actions on proceptivity, the parent compound MTII has also been demonstrated to induce arousal in rats using the laser doppler method with FFT analysis of slow oscillatory activity (Allers et al. 2010d).

In male rats, a similar study demonstrates that following administration of bremelanotide cfos activation occurs within the PVN of the hypothalamus (Molinoff et al. 2003). To assess if this is a potential pathway in females, further investigation was undertaken in naturally cycling estrus rats. Pseudorabies virus (PRV) injection to the clitoris and vagina resulted in transsynaptic labeling present in both the PVN and the MPOA. Furthermore, neurons that were double-labeled for PRV and the melanocortin 4 receptor were found in both the PVN and MPOA, indicating melanocortin pathways exist from both of these regions to the genitalia (Gelez et al. 2010). Between 4 and 8% of the PRV labeled neurons were triplelabeled for both the melanocortin 4 receptor and oxytocin. These data demonstrate direct pathways from both the PVN and the MPOA to the genitalia that could be part of bremelanotide's mechanism of action through activation of melanocortin 4 receptors on oxytocin neurons.

#### **4.3 Flibanserin**

84 Sexual Dysfunctions – Special Issue

metestrus and estrous animals which had effects in this model are the same as those in the

An electrophysiological study was undertaken to further understand the underlying mechanisms for the observed differences in apomorphine actions. Neurons from the paraventricular nucleus, a nucleus important for sexual function and hormonally regulated, were studied in animals from all four stages of the estrous cycle. Firing rates from this nucleus varied substantially across the estrous cycle, with metestrous rates being the highest. In addition subpopulations of neurons were identified: slow neurons which increase in response to apomorphine, and fast neurons which decrease in response to the drug. A greater proportion of the fast neurons were evident in metestrous, accounting for the higher mean firing rate, and leading to relatively greater decreases upon apomorphine

Taken together, these data suggest that during metestrous, a neurological 'stop' signal has been physiologically delivered to the animal which is reflected the animal's behavioral disinterest in a sexual partner. This 'stop' signal is sensitive to, and can be reversed by apomorphine administration. This reversal manifests as increased interest in a sexual partner and increased sexual arousal. An underlying mechanism may be the inhibition of a

The peptide -melanocyte stimulating hormone (MSH) is a product of the proopiomelanocortin pro-hormone. This peptide has long been known to be involved in regulation of energy homeostasis and has been suggested as a target for number medical

In the mid-1980s, a group at the University of Arizona synthesized two highly potent MSH analogues (Hadley et al. 1998). One compound, deemed Melanotan I (MTI) was licensed out and further characterised for utility as a tanning drug, given the known role of MSH in pigmentation. A further analogue, Melanotan II (MTII) was developed which was smaller and the hope was that this would aid in its absorption and tissue distribution. The investigator decided to assess for himself whether this second analogue had the tanning capability seen with MTI and proceeded to dose himself. While it is unclear whether he did achieve a tan, what the investigator reports was an "unrelenting" erection lasting 8 hours. Not long after, this compound was licensed out for further development as a sexual dysfunction treatment candidate. PT-141 is the active metabolite of MTII and ultimately

Clincal trials in women have demonstrated that bremelanotide increases sexual desire and arousal in women with arousal disorders (Diamond et al. 2004; Diamond et al. 2006; Safarinejad 2008). In one study, using vaginal plethysmography to assess vasocongestion, even though subjective scores were increased over placebo, there was no change in vasocongestion measures as compared to controls, confirming that vaginal vasocongestion

Prior to the discovery and development of bremelanotide, melanocortin receptors were not considered to be of great interest within the sexual medicine field. Since that time, a surge of interest has appeared, and along with it a boost in scientific research investigating the

Bremelanotide is an agonist at melanocortin 3 and 4 (MC3 and MC4) receptors, whose primary localisation is in the hypothalamic regions of the brain (Molinoff et al. 2003). The

fast-firing population of neurons within the paraventricular nucleus of the brain.

became the drug development lead compound and was renamed bremelanotide.

is not a suitable method for assessing efficacy of compounds (Diamond et al. 2006).

partner-preference model.

(Richards et al. 2010).

**4.2 Bremelanotide** 

indications (Hedlund 2004).

mechanisms involved.

Flibanserin was discovered in 1990 as part of a program investigating targets for depression (Borsini et al. 1997). This compound was developed based on a very sound rationale for why agonist activity at post-synaptic 5-HT1A receptors and antagonism of 5-HT2A receptors combined would be beneficial to patients with major depressive disorder (Borsini et al. 2002). Unfortuantely, during Phase II trials for depression, flibanserin was not superior to the positive control, paroxetine (Kennedy 2010).

Within the Phase II trials, patients were given the ASEX questionnaire to assess sexual function. Flibanserin treatment improved sexual function in 70% of the patients. Development of flibanserin for depression was discontinued but restarted development for the indication of hypoactive sexual desire disorder. Following several Phase III clinical trials with flibanserin in premenopausal women, the data indicating increased desire and decreased distress following chronic flibanserin treatment is substantial (Clayton et al. 2009; Goldfischer et al. 2009; Jolly et al. 2009).

Following the reassignment of this drug, the search for a mechanism of action began. Within sexual function research, 5-HT1A receptor agonism has long been known to reduce sexual behavior (Ahlenius et al. 1989; Mendelson et al. 1986; Uphouse et al. 1991). The finding that a 5-HT1A agonist is prosexual in women was puzzling. Treatment of rats in pacing chambers either at full estrous priming or sub-hormonal priming also indicated that rats increased proceptive behaviors with chronic flibanserin treatment, and hence, sexual motivation – eliminating the possibility of a species difference in 5-HT1A actions (Allers et al. 2010b; Greggain et al. 2010). Acute dosing of flibanserin has no effect on sexual behavior in rodents.

In pair-bonded marmosets, a study was conducted to compare the effects of chronic flibanserin treatment with that of a commonly used 5-HT1A agonist, 8-OH-DPAT. In this study, flibanserin induced increased affiliative behavior in both pair mates, although only

Paradigms for Preclinical Investigations

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Abstracts595

Med.7310881103

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133346617711773

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Allers, K. A., Gelez, H., Sommer, B., and Giuliano, F.2010bEffects of flibanserin on appetitive

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the females were treated. 8-OH-DPAT induced increased aggression between pairmates after treatment of females alone (Aubert et al. 2009). The result is that flibanserin is clearly not a typical 5-HT1A receptor agonist, but has unique properties that contribute to its mechanism of action.

A key to the difference in flibanserin pharmacology is it's ability to act only at post-synaptic 5-HT-1A receptors. A study by Marazziti et al. demonstrated that in human brain, flibanserin has low nanmolar potency at 5-HT1A receptors in the prefrontal cortex, but none at 10M in the dorsal raphe nucleus (Marazziti et al. 2002). The 5-HT1A receptors in the dorsal raphe nucleus are responsible for regulating serotonin release throughout the brain and typical 5-HT1A agonists will inhibit this release. Post-synaptic receptors are located outside of the dorsal raphe nucleus and have many different functions, including regulating release of all monoamines. To determine how flibanserin administration affects monoamine release two microdialysis studies were undertaken. The first investigated acute dosing while measuring serotonin, dopamine, and norepinephrine in three regions of the brain: the prefrontal cortex, the dorsal raphe, and the hippocampus (Invernizzi et al. 2003). The surprising result was that flibanserin administration decreased serotonin in the prefrontal cortex and dorsal raphe, but not the hippocampus. Recent evidence suggests that hippocampal serotonin is regulated primarily by presynaptic 5-HT1A receptors within the dorsal raphe, so the interpretation for this study is that by acting only at post-synaptic receptors flibanserin can affect serotonin release in selected brain areas. The following microdialysis study indicated that dopamine and norepinephrine are also affected in regionally selective patterns upon chronic dosing (Allers et al. 2010a). Potentially the most significant finding was that within the prefrontal cortex, an area important for general arousal and motivation, basal levels of dopamine and norepinephrine were increased selectively in the prefrontal cortex out of the regions studied (prefrontal cortex, nucleus accumbens, hypothalamic medial preoptic area).

HSDD patients have been shown to have altered cortical reactivity and demonstrate differences in attending to sexual cues (McCall et al. 2006; Vardi et al. 2009). Flibanserin, by increasing two neurotransmitters known to be involved in sexual desire, selectively in regions responsible for attention and awareness, may act to restore these functions (Stahl et al. 2011).
