Natural steroidal chemosignals active in human nasal receptors

In the early 1990s, we reported that naturally occurring steroidal molecules in humans androsta-4,16-dien-3-one (ER670, PH56 or androstadienone (ADO)) and estra-1,3,5,(10,16-tetraen-3-ol (ER830, PH78 or estratetraenol (ETE)), administered in concentrations below olfactory threshold can induce depolarization of the local electrogram recorded from the nasal chemosensory mucosa in human subjects.Reference Monti and Grosser¹ We called these naturally occurring molecules “putative pheromones.” In pharmacology in vitro studies using isolated living human nasal chemosensory cells, ADO and ETE induced robust transient calcium (Ca⁺⁺) membrane currents supporting a membrane (nongenomic) effect of these steroidal compounds.Reference Monti²

In subsequent studies, using an experimental miniprobe that is the extension of a computerized olfactometer for local and topical administration of volatile substances while simultaneously recording the local electrogram from receptors (EGNR) in the nasal chemosensory mucosa,Reference Monti and Grosser^1, Reference Monti³ we reported a rapid depolarizing effect of odorless steroids ADO and ETE on the nasal electrogram of human volunteers. This rapid nongenomic effect was followed by rapid activation of autonomic nervous system (ANS) reflexes and subtle behavioral changes that were distinct for ADO and ETE.Reference Monti and Grosser^1, Reference Monti, Jennings-White, Dolberg and Berliner^4, Reference Grosser, Monti, Jennings-White and Berliner⁵

ADO and ETE are inactive when administered systemically. In a pharmacokinetic study in human volunteers, ADO administered intranasally at 1-hour intervals during 12 consecutive hours was not detected in plasma samples collected at hourly intervals during dosing (HPLC [high-performance liquid chromatography]-mass-mass, assay sensitivity = 2.857 ng/mL).Reference Bustillos-Ventura and Mancera⁶ Furthermore, intranasal and systemic administration of ADO and ETE to laboratory animals (rodent, lagomorph, canid, swine) in doses 100-fold higher than the dose to use in clinical studies did not induce any behavioral or ANS effects. It was concluded that odorless ADO and ETE induced species-specific pharmacological effects through activation of nasal chemosensory cells.Reference Grosser, Monti, Jennings-White and Berliner^5, Reference Monti, Jennings-White and Berliner⁷

Later independent contributions to this field confirmed similar ANS changes, subtle psychological effects, and distinct activation of the hypothalamus (HYP) measured with PET, after intranasal administration of ADO and ETE to human volunteers.^8–19

Other reports showed non–sex-specific effects of putative pheromones ADO and ETE influencing the perception of emotional stimuli in human volunteers.^20–22 The non–sex-dimorphic effects of ADO and ETE were recently questioned in work using these steroidal molecules at high concentrations,Reference Ye, Zhuang, Smeets and Zhou¹⁹ but this is not supported by previous publications using ADO and ETE in concentrations below the olfactory threshold.Reference Monti^3, Reference Grosser, Monti, Jennings-White and Berliner⁵ In a recent article,Reference Banner, Gabay and Shamay-Tsoory²³ men with “high social anxiety” (mean LSAS [Liebowitz Social Anxiety Scale] score of 53.2) reported increased sensitivity to threat and avoidance after nasal delivery of ADO. The adverse effects of ADO could be explained by the high concentration used (above olfactory threshold), although the authors masked ADO odor with eugenol. These results are not consistent with the positive effects of low doses of odorless ADO (below olfactory threshold and without using odor masking) reported in women volunteers.Reference Monti^3, Reference Grosser, Monti, Jennings-White and Berliner⁵ Also, the “high social anxiety” subjects had lower baseline LSAS scores than those required (LSAS ≥ 60) in studies of the therapeutic effect of intranasally administered pherines in subjects with social anxiety disorder (see next section: mean LSAS on entrance was 97.9).Reference Liebowitz, Salman, Nicolini, Rosenthal, Hanover and Monti^24, Reference Liebowitz, Hanover, Draine, Lemming, Careri and Monti²⁵

Pherine molecules

Our preliminary findings led to the development of a new class of more potent neuroactive steroids, focusing on the following advantages: (a) 100% availability of ligand at the peripheral receptor sites immediately after intranasal administration, (b) ultralow dose needed to induce pharmacological effects due to direct administration to the receptor sites, and (c) fast oligosynaptic neural paths from nasal receptors to basal forebrain areas contributing to rapid onset of effect.

The new synthetic neuroactive steroidsFootnote ^a were odorless and specifically designed and formulated to engage human nasal chemosensory receptors (nongenomic effect), looking for rapid and more potent behavioral and ANS effects than their naturally occurring predecessors. The substances were screened in vitro and in vivo, and those without toxicity in laboratory animal studies and lacking binding affinity to steroidal hormone receptors were studied in human volunteers for profiling their pharmacological effects on nasal receptors, ANS reflexes, and psychological effects and to gain information about their possible therapeutic indication. Neuroactive steroids meeting the above profile were included in a new family of therapeutic pharmaceuticals that we called pherines. Reference Monti, Jennings-White, Dolberg and Berliner⁴

Pherines are formulated in a water-based excipient for intranasal administration in spray form using a metered spray pump. Low microgram quantities of pherine administered locally and topically to the surface of the nasal chemosensory mucosal lining produce robust, dose-dependent depolarization of the electrogram (mass receptor potential) ENRG (Figure 1) followed by selective and dose-dependent brain activation of a behavioral and ANS response.Reference Monti, Jennings-White, Dolberg and Berliner^4, Reference Monti, Jennings-White and Berliner^7, Reference Liebowitz, Salman, Nicolini, Rosenthal, Hanover and Monti^24, Reference Liebowitz, Hanover, Draine, Lemming, Careri and Monti^{25, 27–29}

Figure 1.

(A) Representative electrogram (EGNR) traces recorded from the surface of the dorsomedial chemosensory mucosa of the nasal septum in a young adult male volunteer during local administration of control (0.0) and different doses of PH94B. (B) Dose-dependent relationships of PB94B nasal spray on the amplitude of the EGNR in clinically healthy male and female subjects (n = 20). ED₅₀ = .4 μΜ; Hill coefficient = 1. The EGNR was recorded using the Multifunctional Miniprobe (MM®), which is an extension of a computer-driven olfactometer. The therapeutic dose range to use in clinical studies was obtained from the dose–response relationships of PH94B.

Pharmacokinetic studies in human volunteers, administering to the nasal receptor area different doses of androsta-4,16-dien-3β-ol (PH94B or Aloradine), a neuroactive steroid from the androstane family of pherines, produced dose-dependent and reversible activation of the EGNR (ED₅₀ = .4 μM; Hill Coefficient = 1). The dose–response relationships were used to find the effective dose range to administer in clinical studies (Figure 1).

An in vitro pharmacology studyReference Winegar and Monti-Bloch³⁰ in primary cultures of isolated human nasal chemosensory cells using the radiometric Ca⁺⁺ indicator Fura-2 showed significantly increased intracellular Ca⁺⁺ in response to PH94B, and this effect was dose dependent (ED₅₀ = 1.0 μM) and similar to that reported for other neuroactive steroidal compounds acting on nasal receptors.Reference Zhang, De La Cruz, Pinto, Nicolae, Firenstein and Gilad³¹

Activation of nasal receptors by PH94B was followed by decreased sympathetic tone (assessed using physiologic sinus arrhythmia), decreased cardiac and respiratory rate, decreased frequency of electrodermal activity events and electromyogram, and increased body core temperature.Reference Monti, Jennings-White, Dolberg and Berliner⁴ In a separate study, intranasal PH94B also induced specific and dose-dependent activation of brain areas that was different from control and from the effect of primary odors (Figure 2).

Figure 2.

Selective and dose-dependent brain activation induced by odorless pherine PH94B (A) is different from control (SHAM) and brain activation induced by primary odors shown in (B). The results are averaged functional MRI images from human healthy volunteers (n = 8). Warmer colors on the color bars correspond to increased brain activation.

In a pharmacokinetic study in healthy volunteers, with PH94B administered intranasally in spray form at 1-hour intervals during 12 consecutive hours, the concentration of the pherine in plasma samples collected at hourly intervals during dosing was below the detection level of the analytical method (HPLC-M-M; assay sensitivity = 2.857 ng/mL).Reference Bustillos, Mancera, Jagannath and Sanchez³²

The behavioral effects of PH94B were assessed in a double-blind placebo-controlled study involving 90 subjects meeting DSM-IV criteria for social anxiety disorder. Subjects underwent two sets of laboratory-based challenges involving public speaking and social interaction.Reference Liebowitz, Salman, Nicolini, Rosenthal, Hanover and Monti²⁴ During the first set (visit 2), all subjects were pretreated with placebo in a single-blind fashion 15 minutes before each challenge. Those demonstrating significant symptoms were brought back a week later (visit 3) and randomized to PH94B or placebo pretreatment, each followed 15 minutes later by second rounds of performance and social challenges. PH94B was significantly more effective than placebo in reducing both performance and social anxiety as rated by subject self-reports and investigator ratings.Reference Liebowitz, Salman, Nicolini, Rosenthal, Hanover and Monti²⁴

A subsequent 4-week double-blind crossover studyReference Liebowitz, Hanover, Draine, Lemming, Careri and Monti²⁵ was conducted to obtain a preliminary estimate of the efficacy of PH94B when used in real-world situations for a longer period of time. Subjects meeting DSM-IV criteria for social anxiety disorder (n = 22) were randomized to use 1.6 to 3.2 microgram (μg) intranasal PH94B or placebo on an as-needed basis 15 minutes before confronting stressful performance or social situations in their daily life for 2 weeks, after which they were crossed over to the opposite treatment for an additional 2 weeks. Despite the small sample, PH94B demonstrated significant treatment efficacy in several ways. On the primary outcome measure, subjects with marked social anxiety disorder experienced significantly less peak anxiety during social and performance events in their daily lives when using PH94B than when pretreated with placebo (Effect Size = .658, r = .832).Reference Liebowitz, Hanover, Draine, Lemming, Careri and Monti²⁵ Also, between-groups comparisons for the first 2 weeks of treatment showed effect sizes in favor of PH94B on the LSAS total score (effect size = .812) and LSAS avoidance subtotal score (effect size = 1.078), and significantly more subjects rated themselves as treatment responders on the Patient Global Improvement evaluation. What was particular noteworthy is that the degree of improvement seen with PH94B compared to placebo on the LSAS seen in this trial after 2 weeks was comparable in magnitude to that seen after 12 weeks with Food and Drug Administration (FDA) approved medications for Social Anxiety Disorder such as paroxetineReference Stein, Liebowitz, Lydiard, Pitts, Bushnell and Gergel³³ and sertraline.Reference Liebowitz, DeMartinis and Weihs³⁴ These findings extend those of the study that was based on laboratory challenges and shows intranasal PH94B efficacy in real-life situations.

In vitro pharmacology studies using isolated living human nasal chemosensory cells show that PH10 (pregn-4-en-20-yn-3-one), a neuroactive steroid pherine molecule from the pregnane family, induces significant dose-dependent inward membrane currents (ED₅₀ = .2 μM, Hill Coefficient = 1),Reference Monti, Garity and Murray³⁵ and significant dose-dependent depolarization of the electrogram (ENRG) recorded from the nasal chemosensory mucosa in men and women volunteers. The ERG response is followed 15 minutes after intranasal administration of 3.2 μg PH10 by increased plasma NE, 5-HT, and DA, increased sympathetic nervous system tone and frequency of electrodermal activity events.Reference Monti, Diaz Sanchez and Schapper²⁷

In a more recent placebo-controlled, parallel group dose ranging trial in 30 adults with major depressive disorder intranasal PH10 (low dose: 3.2 μg/day and high dose: 6.4 μg/day) reduced Hamilton Depression scores substantially more than did placebo.Reference Monti, Nicolini, Liebowitz and Hanover²⁸ The effect size at the end of the 8-weeks trial was: Effect Size_{High Dose vs Placebo} = .95 and Effect Size Low Dose vs Placebo = .74), suggesting rapid antidepressant activity. Drug–placebo separation appeared during the first week of treatment (Effect Size_{Low Dose vs Placebo} = .72, and Effect Size_{High Dose vs Placebo} = 1.01).