CNS Spectrums Neural circuits of anxiolytic and antidepressant

www.cambridge.org/cns
pherine molecules
Louis Monti1* and Michael R. Liebowitz2,3
1
Pherin Pharmaceuticals, Inc., Mountain View, California, USA, 2The Medical Research Network, LLC, New York, USA,
Review and 3Department of Psychiatry, College of Physicians and Surgeons, Columbia University, New York, USA
Cite this article: Monti L, and Liebowitz MR
(2022). Neural circuits of anxiolytic and Abstract
antidepressant pherine molecules. CNS
Spectrums 27(1), 66–72. In this review, we describe proposed circuits mediating the mechanism of action of pherines,
https://doi.org/10.1017/S109285292000190X a new class of synthetic neuroactive steroids with demonstrated antianxiety and antidepres-
sant properties, that engage nasal chemosensory receptors. We hypothesize that afferent
Received: 02 April 2020
signals triggered by activation of these peripheral receptors could reach subgroups of olfactory
Accepted: 06 October 2020
bulb neurons broadcasting information to gamma-aminobutyric acid (GABAergic) and
corticotropin-releasing hormone (CRH) neurons in the limbic amygdala. We propose that
chemosensory inputs triggered by pherines project to centrolateral (CeL) and centromedial
Author for correspondence: (CeM) amygdala neurons, with downstream effects mediating behavioral actions. Anxiolytic
*Louis Monti, MD, PhD, pherines could activate the forward inhibitory GABAergic neurons that facilitate the release of
Email: [email protected]
neuropeptide S (NPS) in the locus coeruleus (LC) and GABA in the bed nucleus of the stria
terminalis (BNST) and inhibit catecholamine release in the LC and ventral tegmental area (VTA)
leading to rapid anxiolytic effect. Alternatively, antidepressant pherines could facilitate the CRH
and GABAergic neurons that inhibit the release of NPS from the LC, increase glutamate release from
the BNST, and increase norepinephrine (NE), dopamine (DA), and serotonin release from the LC,
VTA, and raphe nucleus, respectively. Activation of these neural circuits leads to rapid antidepres-
sant effect. The information provided is consistent with this model, but it should be noted that some
steps on these pathways have not been demonstrated conclusively in the human brain.

Introduction
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 thresh-
old can induce depolarization of the local electrogram recorded from the nasal chemosensory
mucosa in human subjects.1 We called these naturally occurring molecules “putative phero-
mones.” 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.2
In subsequent studies, using an experimental miniprobe that is the extension of a comput-
erized olfactometer for local and topical administration of volatile substances while simulta-
neously recording the local electrogram from receptors (EGNR) in the nasal chemosensory
mucosa,1,3 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.1,4,5
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).6 Furthermore,
intranasal and systemic administration of ADO and ETE to laboratory animals (rodent, lago-
© The Author(s), 2020. Published by Cambridge
morph, canid, swine) in doses 100-fold higher than the dose to use in clinical studies did not induce
University Press. This is an Open Access article, any behavioral or ANS effects. It was concluded that odorless ADO and ETE induced species-
distributed under the terms of the Creative specific pharmacological effects through activation of nasal chemosensory cells.5,7
Commons Attribution licence (http:// Later independent contributions to this field confirmed similar ANS changes, subtle psycho-
creativecommons.org/licenses/by/4.0/), which logical effects, and distinct activation of the hypothalamus (HYP) measured with PET, after
permits unrestricted re-use, distribution, and
reproduction in any medium, provided the
intranasal administration of ADO and ETE to human volunteers.8–19
original work is properly cited. 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,19 but this is not supported by previous publications using
ADO and ETE in concentrations below the olfactory threshold.3,5 In a recent article,23 men with

https://doi.org/10.1017/S109285292000190X Published online by Cambridge University Press

 CNS Spectrums 67

A B
PH94B (µg)
0.0
0.4
0.8

Mean EGNR (-mV)

1.6

3.2

6.4

- 10 mV
12.8
PH94B (µg)

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.

“high social anxiety” (mean LSAS [Liebowitz Social Anxiety Scale] to the surface of the nasal chemosensory mucosal lining produce
score of 53.2) reported increased sensitivity to threat and avoidance robust, dose-dependent depolarization of the electrogram (mass
after nasal delivery of ADO. The adverse effects of ADO could be receptor potential) ENRG (Figure 1) followed by selective and
explained by the high concentration used (above olfactory thresh- dose-dependent brain activation of a behavioral and ANS
old), although the authors masked ADO odor with eugenol. These response.4,7,24,25,27–29
results are not consistent with the positive effects of low doses of Pharmacokinetic studies in human volunteers, administering to
odorless ADO (below olfactory threshold and without using odor the nasal receptor area different doses of androsta-4,16-dien-3β-ol
masking) reported in women volunteers.3,5 Also, the “high social (PH94B or Aloradine), a neuroactive steroid from the androstane
anxiety” subjects had lower baseline LSAS scores than those family of pherines, produced dose-dependent and reversible acti-
required (LSAS ≥ 60) in studies of the therapeutic effect of intra- vation of the EGNR (ED50 = .4 μM; Hill Coefficient = 1). The dose–
nasally administered pherines in subjects with social anxiety disor- response relationships were used to find the effective dose range to
der (see next section: mean LSAS on entrance was 97.9).24,25 administer in clinical studies (Figure 1).
An in vitro pharmacology study30 in primary cultures of isolated
human nasal chemosensory cells using the radiometric Ca++ indi-
Pherine molecules
cator Fura-2 showed significantly increased intracellular Ca++ in
Our preliminary findings led to the development of a new class of response to PH94B, and this effect was dose dependent (ED50 =
more potent neuroactive steroids, focusing on the following advan- 1.0 μM) and similar to that reported for other neuroactive steroidal
tages: (a) 100% availability of ligand at the peripheral receptor sites compounds acting on nasal receptors.31
immediately after intranasal administration, (b) ultralow dose needed Activation of nasal receptors by PH94B was followed by decreased
to induce pharmacological effects due to direct administration to the sympathetic tone (assessed using physiologic sinus arrhythmia),
receptor sites, and (c) fast oligosynaptic neural paths from nasal decreased cardiac and respiratory rate, decreased frequency of elec-
receptors to basal forebrain areas contributing to rapid onset of effect. trodermal activity events and electromyogram, and increased body
The new synthetic neuroactive steroidsa were odorless and core temperature.4 In a separate study, intranasal PH94B also induced
specifically designed and formulated to engage human nasal che- specific and dose-dependent activation of brain areas that was dif-
mosensory receptors (nongenomic effect), looking for rapid and ferent from control and from the effect of primary odors (Figure 2).
more potent behavioral and ANS effects than their naturally occur- In a pharmacokinetic study in healthy volunteers, with PH94B
ring predecessors. The substances were screened in vitro and in administered intranasally in spray form at 1-hour intervals during
vivo, and those without toxicity in laboratory animal studies and 12 consecutive hours, the concentration of the pherine in plasma
lacking binding affinity to steroidal hormone receptors were stud- samples collected at hourly intervals during dosing was below the
ied in human volunteers for profiling their pharmacological effects detection level of the analytical method (HPLC-M-M; assay sensi-
on nasal receptors, ANS reflexes, and psychological effects and to tivity = 2.857 ng/mL).32
gain information about their possible therapeutic indication. Neu- The behavioral effects of PH94B were assessed in a double-blind
roactive steroids meeting the above profile were included in a new placebo-controlled study involving 90 subjects meeting DSM-IV
family of therapeutic pharmaceuticals that we called pherines.4 criteria for social anxiety disorder. Subjects underwent two sets of
Pherines are formulated in a water-based excipient for intrana- laboratory-based challenges involving public speaking and social
sal administration in spray form using a metered spray pump. Low interaction.24 During the first set (visit 2), all subjects were pretreated
microgram quantities of pherine administered locally and topically with placebo in a single-blind fashion 15 minutes before each chal-
lenge. Those demonstrating significant symptoms were brought
a
For the definition of neuroactive steroids, see Reference 26. back a week later (visit 3) and randomized to PH94B or placebo

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 68 L. Monti and M. R. Liebowitz

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.

pretreatment, each followed 15 minutes later by second rounds of that was based on laboratory challenges and shows intranasal
performance and social challenges. PH94B was significantly more PH94B efficacy in real-life situations.
effective than placebo in reducing both performance and social In vitro pharmacology studies using isolated living human nasal
anxiety as rated by subject self-reports and investigator ratings.24 chemosensory cells show that PH10 (pregn-4-en-20-yn-3-one), a
A subsequent 4-week double-blind crossover study25 was con- neuroactive steroid pherine molecule from the pregnane family,
ducted to obtain a preliminary estimate of the efficacy of PH94B induces significant dose-dependent inward membrane currents
when used in real-world situations for a longer period of time. (ED50 = .2 μM, Hill Coefficient = 1),35 and significant dose-
Subjects meeting DSM-IV criteria for social anxiety disorder dependent depolarization of the electrogram (ENRG) recorded
(n = 22) were randomized to use 1.6 to 3.2 microgram (μg) intra- from the nasal chemosensory mucosa in men and women volun-
nasal PH94B or placebo on an as-needed basis 15 minutes before teers. The ERG response is followed 15 minutes after intranasal
confronting stressful performance or social situations in their daily administration of 3.2 μg PH10 by increased plasma NE, 5-HT, and
life for 2 weeks, after which they were crossed over to the opposite DA, increased sympathetic nervous system tone and frequency of
treatment for an additional 2 weeks. Despite the small sample, electrodermal activity events.27
PH94B demonstrated significant treatment efficacy in several ways. In a more recent placebo-controlled, parallel group dose rang-
On the primary outcome measure, subjects with marked social ing trial in 30 adults with major depressive disorder intranasal
anxiety disorder experienced significantly less peak anxiety during PH10 (low dose: 3.2 μg/day and high dose: 6.4 μg/day) reduced
social and performance events in their daily lives when using Hamilton Depression scores substantially more than did placebo.28
PH94B than when pretreated with placebo (Effect Size = .658, The effect size at the end of the 8-weeks trial was: Effect SizeHigh Dose
r = .832).25 Also, between-groups comparisons for the first 2 weeks vs Placebo = .95 and Effect Size Low Dose vs Placebo = .74), suggesting
of treatment showed effect sizes in favor of PH94B on the LSAS rapid antidepressant activity. Drug–placebo separation appeared
total score (effect size = .812) and LSAS avoidance subtotal score during the first week of treatment (Effect SizeLow Dose vs Placebo = .72,
(effect size = 1.078), and significantly more subjects rated them- and Effect SizeHigh Dose vs Placebo = 1.01).
selves 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 Body
seen in this trial after 2 weeks was comparable in magnitude to that
seen after 12 weeks with Food and Drug Administration (FDA) Olfactory neural circuits
approved medications for Social Anxiety Disorder such as parox- Traditionally, it is accepted that in most mammals, olfaction is
etine33 and sertraline.34 These findings extend those of the study accomplished by two subsystems: main olfactory system (MOS)

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 CNS Spectrums 69

broadcasting sensory inputs from odor chemosignals to the Furthermore, neural inputs from the BLA reach GABAergic–
main olfactory bulbs (OB) projecting to the olfactory tubercle, PKCδ-negative ON-neurons in the CeL. It has been reported that
piriform cortex, medial amygdala (MeA), and cortical amygdala CeL outputs via an intercalated feed-forward series of GABAergic
(CA)36,37; and the accessory olfactory system (AOS) conveying interneurons and also through CRH neurons can stimulate gluta-
pheromone chemosignals to the accessory olfactory bulbs matergic neurons in the BNST oval area and in the prefrontal cortex,
(AOB), which in turn reach basolateral amygdala (BLA) neurons with concurrent stimulation of NE, DA, and serotonin release from
that project to the anterior and ventromedial HYP, bed nucleus the midbrain (LC, VTA, and RN), (Figure 3). Activation of these
of the stria terminalis (BNST), medial preoptic area (MPA), circuits leads to simultaneous inhibition of GABAergic neurons in the
striatum (ST), locus coeruleus (LC), parabrachial nucleus BNST and NPS-releasing neurons in the LC, increased sympathetic
(PN), and prefrontal cortex (PFC).38,39 Activation of these neu- system tone, and a neuroendocrine response,53,60–62 all in agreement
ral circuits is involved in the modulation of different social with the neurocircuits of mood disorders.63
behaviors.
More recently, it was reported that, in mammals, there are
subsets of OB neurons that share synaptic connections in the same Proposed mechanisms of pherine activity
limbic amygdala nuclei as the AOS, and olfactory activity behaviors
originally assigned to the AOS are mediated through the main We hypothesize that the PH94B-induced rapid decrease (latency ≤
olfactory epithelium and the MOS.40–48 Therefore, since humans 400 ms) of sympathetic tone64 and rapid improvement (latency = 10-
do not have an identifiable AOB, we hypothesize that this could be 15 minutes) in performance anxiety and social interaction anxiety24,25
the neural path by which chemosensory inputs triggered by pher- are triggered by sensory inputs originating in nasal chemosensory
ines could reach the amygdala nuclei. neurons that stimulate subset of OB neurons projecting to the MeA
Numerous studies show that the MeA, a key structure in the and BLA. There is evidence that MeA and BLA neurons trigger the
control of social behavior, projects to GABAergic neurons in the forward inhibitory GABAergic–PKCδ-positive OFF-neurons in the
centrolateral amygdala (CeL) that trigger the forward inhibitory CeL and CeM amygdala, which downstream effects mediating
GABAergic circuits directly mediating fear and anxiety and also to behavioral actions that directly mediate social behavior, fear, and
the BNST and the HYP involved in the regulation of innate defense anxiety51,52,56,63,64 (Figure 3). The modulation of neural circuits
responses49–51 (Figure 3). involved in the pathogenesis of social anxiety disorder55–57,59,65–68
It has been shown that olfactory projections to the cortical appears to be consistent with the PH94B-induced acute anxiolytic
amygdala can also trigger BLA neurons52 which synapse with the effects and autonomic nervous system changes reported in our
important contingent of GABAergic forward inhibitory neurons in clinical studies in patients diagnosed with social anxiety disorder.24,25
the lateral (CeL) and medial (CeM) division of the central amygdala We also hypothesize that the rapid change in sympathetic tone
involved in the modulation of fear and anxiety.42,44,48,49,53–56 More and dose-dependent improvement in Hamilton Depression scores
recent evidence shows that GABAergic-PKCδ-positive OFF- (HAM-D) scores induced after intranasal administration of PH1028
neurons in the CeL facilitate the release of neuropeptide S (NPS) are the result of activation of glutamatergic neurons in the BLA that
in the LC and GABA from anterolateral BNST through forward in turn trigger the GABAergic–PKCδ-negative ON-neurons in the
inhibition of GABAergic neurons in the CeM, and there is concur- CeL. The antidepressant effect of PH10 through activation of nasal
rent inhibition of NE, DA, and 5-HT release from the midbrain and chemosensory receptors is supported by other independent studies
decreased sympathetic system tone through inhibition of neurons showing an important association of the olfactory system and mood
in the posterior HYP.42,51,53–61 disorders.63,68–71

Figure 3. Schematic diagram showing the olfactory connections to the limbic amygdala and related areas. The olfactory bulb (OB) connections to the limbic amygdala are shorter
and bypass the thalamus thus being a fast (shorter latency) neural input to the basal forebrain compared to other sensory afferent systems.
Abbreviations: BLA, basolateral amygdala; BNST, bed nucleus of the stria terminalis; CA, cortical amygdala; CeA, central amygdala; CeL, centrolateral amygdala; CeM, centromedial
amygdala; HYP, hypothalamus; LC, locus ceruleus; MeA, medial amygdala; OB, olfactory bulb; PFC, prefrontal cortex; RN, raphe nucleus; THAL, thalamus; VTA, ventral tegmental
area.

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 70 L. Monti and M. R. Liebowitz

Conclusions Disclosures. Dr. Monti reports financial support from Pherin Pharmaceuti-
cals (employed as President and owns stock from Pherin Pharmaceuticals and
There is extensive evidence showing the human olfactory system’s VistaGen Therapeutics). In addition, Dr. Monti has patents issued for Treat-
role in social behavior, food ingestion, appetite regulation, awareness ment of Social Phobia (US Patent 8,309,539) and for Treatment of Depressive
of the surrounding environment, and detection of hazards.72,73 Disorders (US Patent 10,322,138).
Unlike other sensory systems, olfactory inputs do not have a synaptic Dr. Liebowitz reports financial support from Pherin Pharmaceuticals (stock
relay in the thalamus to be routed to the cortex. Rather, they are options) and from VistaGen Therapeutics (stock options and consulting fees).
wired to the limbic amygdala, HYP, and hippocampus, which pro- VistaGen Therapeutics has licensed PH94B and PH10 from Pherin Pharma-
vides olfaction with a unique and potent power to influence mood, ceuticals.
acquisition of new information, and its use in many different con-
texts including social interaction, fear, emotions, and the memory
components of behavior.74–80
Thus, it is reasonable to assume that in humans, there are References
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