Novel investigational therapeutics for panic disorder.

Review

Novel investigational therapeutics for panic disorder

Expert Opin. Investig. Drugs Downloaded from informahealthcare.com by Nyu Medical Center on 01/13/15 For personal use only.

Giampaolo Perna†, Koen Schruers, Alessandra Alciati & Daniela Caldirola †

1.

Introduction

2.

Animal models of panic

3.

Preclinical and clinical studies

4.

Conclusion

5.

Expert opinion

Hermanas Hospitalarias - Villa San Benedetto Menni Hospital, FoRiPsi, Department of Clinical Neurosciences, Como, Italy

Introduction: Panic disorder (PD) is a common disabling anxiety disorder associated with relevant social costs. Effective anti-panic medications exist but have several drawbacks. From a clinical perspective, there is still a strong unmet need for more effective, faster acting and more tolerable therapeutic treatments. Areas covered: The authors review the available results on novel mechanismbased anti-panic drugs that are under investigation in animal studies up to Phase II studies. The preclinical studies investigated include: the modulators of the glutamate/orexin/cannabinoid systems, corticotrophin-releasing factor 1 (CRF1)/arginine vasopressine V1B/angiotensin II receptor antagonists and neuropeptide S. The Phase I/II studies investigated include: the modulators of the glutamate system, isoxazoline derivative, translocator protein (18 kDa) ligands and CRF1/neurokinin receptor antagonists. Expert opinion: There has been little progress in recent times. However, glutamate- and orexin-related molecular targets may represent very promising opportunities for treating panic attacks. Very preliminary findings suggest that the antagonists of CRF1 and A-II receptors may have anti-panic properties. However, new medications for PD are far from being implemented in clinical use. The reasons are multiple, including: the heterogeneity of the disorder, the translational validity of animal models and the insufficient use of biomarkers in preclinical/clinical studies. Nevertheless, biomarker-based strategies, pharmacogenomics, ‘personalized psychiatry’ and the NIH’s Research Domain Criteria approach could help to remove those obstacles limiting drug development. Keywords: glutamate, novel drugs, orexin, panic disorder Expert Opin. Investig. Drugs [Early Online]

1.

Introduction

Panic disorder (PD) is associated with high rate of relapse, psychiatric/medical comorbidity, significant impairment of quality of life and relevant social costs. Its lifetime prevalence is 3 -- 4% [1]. Despite several medications have been used for treating PD, including selective serotonin reuptake inhibitors (SSRIs) (first-line pharmacological intervention), serotonin-norepinephrine reuptake inhibitors (SNRIs), tricyclic antidepressants (TCAs) and benzodiazepines (BDZs) [2], a significant proportion of patients does not respond to treatment [3]. Moreover, SSRIs, SNRIs and TCAs induce an initial increase in anxiety and need several weeks to exert their therapeutic effects. Although SSRIs/SNRIs long-term treatment is better tolerated than TCAs, potential side effects include weight gain and sexual dysfunctions. Finally, BDZs exert fast anxiolytic effects but can cause sedation, memory impairment, tolerance and abuse [3]. Approximately 20 -- 40% of subjects with PD do not fully respond to pharmacotherapy. A similar rate does not improve with cognitive behavioral therapy (CBT), and so far, combining CBT to pharmacotherapy has not sufficiently filled this gap. Twenty-five to fifty per cent of patients relapse within 6 months after drug 10.1517/13543784.2014.996286 © 2014 Informa UK, Ltd. ISSN 1354-3784, e-ISSN 1744-7658 All rights reserved: reproduction in whole or in part not permitted

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G. Perna et al.

Article highlights. . .

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There is a strong medical need for novel pharmacologic treatments in panic disorder (PD). Preclinical studies that used the panic-prone rats, a wellvalidated animal model to reproduce panic attacks (PAs), suggest that both metabotropic glutamate II receptor agonists and orexin 1 receptor antagonists may be the most promising compounds for treating PAs. Very preliminary findings suggest that also the antagonists of corticotrophin-releasing factor 1 and A-II receptors may have anti-panic properties. Clinical validation in humans is unsatisfactory and no new compound is close to being ready for clinical use in PD. The use of biomarker-based approaches, pharmacogenomics, ‘personalized psychiatry’ and Research Domain Criteria approach may be a valuable strategy for developing new anti-panic medications.

This box summarizes key points contained in the article.

discontinuation and up to 50% still experience residual panicphobic symptoms. Finally, up to 30% of patients still suffer from a full-blown disorder after 3 -- 6 years [4]. From a clinical perspective, there is still a strong unmet need for more effective, faster acting and more tolerable therapeutic treatments for PD. Many reasons may explain the difficulties to fill these gaps. First, PD is a heterogeneous condition that results from the interplay of unexpected (i.e., occurring out of the blue) panic attacks (PAs), that is, ‘the core symptoms’ of the disorder, and other symptoms following the occurrence of PAs, that is, anticipatory anxiety and phobic behaviors associated with expected PAs (i.e., induced by feared cues, such as situations where PAs have occurred). These clinical phenomena, whose presence can vary across subjects with the disorder, are qualitatively different and probably involve distinct neural circuits [5,6]. In addition, different panic subtypes, based on symptom profiles of PAs, may exist [7,8]. Second, the underlying pathophysiology of PD is still not well understood. Some contemporary theories conceive PAs as primal defensive reactions to threat within the internal milieu of the body, which might be attributable to a misfiring suffocation alarm and/or malfunction of brain circuits modulating defensive responses. Donald Klein’s “Suffocation False Alarm Theory’ was introduced in 1993. It has received criticism but also spawned research into the mechanisms of panic. Its further development postulates abnormal respiratory regulation and/or hypersensitive central carbon dioxide (CO2)/hydrogen ion (H+) chemoreception in both spontaneous and experimentally induced (by CO2 inhalation or sodium lactate [NaLac] infusion) PAs, which are considered qualitatively distinct from fear responses [9-12]. Other authors postulated, in the generation of Pas, a critical role of the midbrain dorsal periaqueductal gray (DPAG) and the hypothalamus, which are brain areas involved in unconditioned defensive reactions to proximal/ interoceptive cues. [6,13,14].. According to both these theories, 2

PAs may be related to the activation of phylogenetically primitive brain structures, which process basic physiological functions, such as brainstem, hypothalamus and insula, whereas anticipatory anxiety/phobic behaviors may be influenced by higher brain systems, such as the prefrontal cortex [15-17]. Conversely, Gorman et al. proposed strong similarities between human PAs and conditioned fear responses in animals, conceiving both PAs and their related phenomena as the results of a dysfunctional ‘fear network’ modulated by the amygdala and the limbic system [18]. However, recent findings did not support this idea but suggested that the amygdala may not play a crucial role in the occurrence of PAs but may be mainly involved in anxiety/conditioned fear behaviors following the PAs [6,17,19]. In line with this, a recent functional MRI study pointed to the importance of brainstem structures, rather than the amygdala, in the mechanisms of panic in humans [16]. Third, several neurotransmitters acting in different CNS areas and influencing each other may be involved in modulating these putative processes. Unfortunately, the mechanisms of action of the existing anti-panic drugs are not completely understood so far [4]. Fourth, it is not clear which animal models can best represent PD in humans [20]. Previous preclinical studies used different animal models based on different theoretical constructs and thus may yield different translational validity [6,20,21]. These circumstances may explain why there have been few therapeutic advances in PD in the near past. In this paper, we overview novel mechanism-based anti-panic drugs that are currently under investigation in animal studies up to Phase II studies. Given the heterogeneity of the psychopathological phenomena that are included in the diagnosis of PD, we focused on drugs that are under investigation for treating PAs, which is the hallmark of PD. 2.

Animal models of panic

In reviewing preclinical studies, we selected those that used animal models of PAs with sufficient translational validity ([6,20,21]). Three validated animal models have been used to test the anti-panic properties of novel compounds under current investigation. In the following paragraphs we describe these models. Chronic disinhibition of the dorsomedial/ perifornical hypothalamus: the panic-prone rats

2.1

Chronically disrupting the inhibitory GABA-mediated activity, by infusion of the GABA synthesis inhibitor L-allyglicine in the dorsomedial/perifornical hypothalamic region (DMH/PeF) of rats, results in a protracted glutamatemediated activation of this area. As a consequence, rats display increased panic-associated behaviors and cardio-respiratory responses to interoceptive stimuli that provoke PAs in humans (panic-prone state) [22]. NaLac infusion in panicprone rats results both in rapid panic-like cardio-respiratory responses (i.e., tachypnea, tachycardia, hypertension) and

Expert Opin. Investig. Drugs (2014) 24(3)



increased anxiety-like behavior in the social interaction (SI) test. This response is similar to that observed in humans with PD during PAs induced experimentally by panicogenic agents (NaLac infusion, CO2 inhalation), and it is not due to a general increased arousal, becauser there are no changes in the baseline acoustic startle response [23]. These features support the face validity of this model, which is the degree to which the model reproduces phenomenological features of PAs [22]. Acute pretreatment with alprazolam, an effective anti-panic drug, prevents NaLac-induced panic in panicprone rats (postdictive validity, i.e., the sensitivity to effective existing drugs) [24], even though the effects of SSRIs/TCAs/ SSNRIs have not been tested yet. The construct validity of this model (i.e., the extent to which the model is related to the neural underpinnings of PAs) is supported by the fact that DMH/PeF circuits coordinate respiratory, autonomic and behavioral responses for a variety of homeostatic mechanisms that are involved in panic/defense responses [22]. Moreover, DMH/PeF communicates with the circumventricular organs (CVOs), a region without blood--barrier that is critical for sensing changes in several plasma parameters, including pH and osmolarity [25]. Via CVOs pathways, subthreshold doses of NaLac in panic-prone rats activate the DMH/PeF, which in turns relays the signal to efferent targets implicated in panic-like responses such as the brainstem regions regulating autonomic/respiratory activity [26]. In addition, NaLac infusion activates the DPAG, which is involved in unconditioned defensive reactions to interoceptive cues and may be a critical locus of the ‘suffocation alarm system’ [17,27]. Disinhibition of the DMH/PeF hyperactivates the hypothalamic orexine (ORX) system, which is involved in hypersensitivity to hypercapnia, a crucial hallmark of panic vulnerability in humans (see Section 3.2) [23]. Finally, during NaLac infusion in panic-prone rats several limbic structures (e.g., the bed nucleus of the stria terminalis [BNST] and the amygdala) are activated, but they do not seem crucial for the panic-like responses. Indeed, pharmacological inactivation of the BSNT hinders anxiety-associated behaviors without altering NaLac-induced cardio-respiratory response, and the experimental disinhibition of the sole BNST results in heightened baseline anxiety, but not in vulnerability to cardio-respiratory responses following NaLac infusion [22,26]. These findings suggest that activation of the limbic structures may be implicated in conditioned fear processes after PAs rather than in PAs per se, and this agrees with the most validated theories of panic [6,10-12,22]. Overall, this preclinical model seems to adequately reflect PAs. The elevated T-maze The elevated T-maze (ETM) assesses two qualitatively different behaviors in rats: conflict-evoked anxiety behaviors (i.e., inhibitory avoidance/risk assessment) and defensive behaviors evoked by unconditioned cues (i.e., escape responses). These behaviors have been related to human generalized anxiety and panic, respectively [20,28]. The ETM consists of one 2.2

enclosed arm (40-cm high walls) perpendicular to two opposed open arms (50 12 cm each), elevated 50 cm from the floor. The enclosed arm is surrounded by 40-cm high walls and is perpendicular to the two opposed open arms. This test is based on the innate fear of height/openness shown by rats. When placed at the end of the enclosed arm, the rat cannot see the open arms until its head is pokes out the walls. Because the open arms are unpleasant, rats will learn inhibitory avoidance (anxiety-like behavior) when they are placed at the end of the enclosed arm. On the other hand, when the animal is placed at the end of one of the open arms it can move towards the enclosed arm, performing an escape response (panic-like behavior). Experimental evidence showed that these distinct responses recruit different neural circuits. Escape response mainly involves the dorsal raphe nuclei-DPAG pathways, that is, circuits that modulate panic/defense responses to unconditioned stimuli and are probably recruited during PAs (construct validity). Accordingly, pretreatment with alprazolam or chronic administration of SSRIs (clinically effective anti-panic drugs) inhibit escape responses, whereas anxiolytic drugs with good clinical effectiveness on generalized anxiety but not on PAs (e.g., buspirone, ritanserin, midazolam) impair inhibitory avoidance acquisition while leaving the escape response unchanged (postdictive validity). Conversely, the face validity of this model is more limited, because the behavioral measure (escape response) does not fully mimic PAs and no objective measure of respiratory/ autonomic responses is included in the model [6,20,28]. The electrical stimulation of the DPAG The model based on the electrical stimulation of the DPAG in rats consists of evaluating behavioral responses following gradual increases in the intensity of an electrical current. Animals are placed inside an arena and the threshold to induce escape responses is recorded by trained observers. The construct and face validity of this model is similar to that described for the ETM (see Section 2.2). Effective anti-panic treatments (e.g., chronic administration of TCAs or SSRIs) increase the escape response threshold, whereas clinically ineffective anti-panic drugs (e.g., buspirone, maprotiline) do not change this threshold [20]. 2.3

3.

Preclinical and clinical studies

In this section, we review preclinical and Phase I or II clinical studies investigating novel drugs with potential anti-panic properties. For each compound, we describe the preclinical studies and the clinical ones. All preclinical and clinical studies are listed in Tables 1 and 2, respectively. Modulators of glutamatergic receptors Glutamate is the main excitatory neurotransmitter in the human CNS that functionally interacts with GABA (the main inhibitory neurotransmitter) [29]. Glutamate receptors encompass ionotropic (AMPA, Kainate, NMDA) and 3.1


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Table 1. Preclinical studies. Compound

Mechanisms of action

Panic model

Effects

Ref.

LY354740 (Eglumegad)

mGlu2/3 receptor agonist

NaLac-induced panic-like response in ‘panic-prone’* rats

[24]

CBiPES, THIIC

mGlu2 receptor agonist


MK-801

NMDA receptor antagonist

SB334867, SB408124

Orexine type 1 receptor antagonist CRF1 receptor antagonist

NaLac-induced panic-like response in ‘panic-prone’* rats NaLac-induced panic-like response in ‘panic-prone’* rats ETM, electrical stimulation of DPAG NaLac-induced panic-like response in ‘panic-prone’* rats NaLac --induced panic --like response in ‘panic-prone’* rats ETM, electrical stimulation of DPAG ETM

Inhibition of NaLac-induced panic-like responsez (similar to alprazolam) Inhibition of NaLac-induced panic-like responsez (similar to alprazolam) Inhibition of NaLac-induced panic-like responsez Inhibition of NaLac-induced panic-like responsez Inhibition of CRF-induced escape behaviors Inhibition of NaLac-induced panic-like responsez Inhibition of NaLac-induced panic-like responsez Reduction of escape behaviors

Antalarmin JNJ19567470/ CRA5626 Losartan, Saralasin Capsazepine URB597 XBD173(AC5216, Emapunil)

CRF1 receptor antagonist Angiotensin II receptor antagonists Transient receptor potential vannilloid type 1 antagonist Inhibitor of AEA hydrolysis by the enzyme FAAH Selective phenylpurine highaffinity TPSO ligand


Spantide TASP0233278

NK-1 receptor antagonist Arginine vasopressin V1B receptor antagonist

Electrical stimulation of DPAG NaLac-induced panic-like response in ‘panic-prone’* rats

NPS

NPS receptor agonist

ETM

Reduction of escape behaviors Inhibition of NaLac-induced anxiety-like behaviors (SI test) (cardio-respiratory parameters not assessed) Reduction of escape behaviors Inhibition of NaLac-induced anxiety-like behaviors (SI). No activity on respiratory response Reduction of escape behaviors and inhibitory avoidance

[33]

[34] [42] [49] [50] [53] [67]; [66] [68] [73]

[82] [83]

[87]

*Panic-prone rats: rats with chronic disinhibition of dorsomedial/perifornical hypothalamus. z Panic-like response: anxiety-like behavior as measured by SI test and rapid increase of cardio-respiratory activity (tachypnea, tachycardia, hypertension). AEA: Anandamide (endogenous opioid receptor ligand); CRF: Corticotrophin-releasing factor; DPAG: Dorsal periaqueductal gray; ETM: Elevated T-maze; FAAH: Fatty acid amide hydrolase; NPS: Neuropeptide S; SI: Social interaction.

Table 2. Clinical studies. Compound

Mechanisms of action

LY354740 (Eglumegad) mGluR2/3 receptor agonist LY354740 (Eglumegad) mGluR2/3 receptor agonist

Study design

Effects

Ref.

Phase II, randomized, doubleblind, PLC-controlled, multi-centre Phase II, randomized, doubleblind, PLC-controlled, multicentre; active comparator = PRX Phase I, randomized, doubleblind, PLC-controlled

LY354740 > PLC in reducing CO2induced PAs after 4 weeks LY354740 = PLC = PRX in primary measure: % of patients without PAs after 9 weeks Significant reduction of general anxiety responses to 7.5% CO2 inhalation (induced PAs not evaluated) XBD173 = alprazolam > PLC in reducing cholecystokinin-4 induced anxiety response Vestipitant > PLC in reduction of anxiety responses (DVAS-A%). Alprazolam > PLC > vestipitant in reduction of panic symptoms list total score. Vofopitant = no effects Aims: clinical efficacy, tolerability. Results: not available

[36]

R317573

CRF1 receptor antagonist

XBD173(AC5216, Emapunil)

Selective phenylpurine high-affinity TPSO ligand

Phase I, PLC-, alprazolamrandomized

GW597599(vestipitant) GR205171 (vofopitant)

NK1 receptor antagonists

Phase I, randomized, doubleblind, PLC-, alprazolam-controlled

ABIO08/01

Inhibitor of calmodulindependent II kinase

Phase II, randomized, doubleblind, PLC-controlled

CRF: Corticotrophin-releasing factor; NK1: Neurokinin 1; PLC: Placebo; PRX: Paroxetine; PAs: Panic attacks; TPSO: Translocator protein (18 kDa).

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[37]

[46]

[73]

[80]

[90]



metabotropic Group I (subtype mGluR1,5), Group II (subtype mGluR2,3) and Group III (subtype mGluR4,6,7,8) receptors [30]. Glutamate modulates the DMH/PeF, DPAG and the orexin system, which are brain pathways involved in panic/ defensive responses [22]. In panic-prone rats, glutamate receptors activation is necessary for panic responses [22]. In addition, glutamate regulates the functions of several neurotransmitters implicated in the pathophysiology of panic, such as serotonin and noradrenaline [30]. In subjects with PD, there is evidence of reduced GABAergic tone. Abnormal GABAA-BDZ receptor binding and GABA levels reductions were found in various brain regions of subjects with PD [31]. Moreover, genetic polymorphisms in genes encoding the glutamate decarboxylases (i.e., enzymes synthesizing GABA from glutamate) were associated with PD [32]. Thus, an unbalance between glutamatergic and GABAergic tone in panicgenerating sites may play a role in PAs, as also shown in panic-prone rats. Therefore, the glutamatergic system may be a promising target for treating PAs. 3.1.1

Preclinical studies Group II-metabotropic glutamate receptors agonists

3.1.1.1

LY354740 is a selective agonist of the Group II metabotropic glutamate receptors (mGluR2/3). Group II mGlu receptors decrease glutamate neurotransmission acting both presynaptically (inhibitory autoreceptors) and postsinaptically [24]. Group II mGlu receptors are widely distributed across several brain regions involved in panic mechanisms, such as DMH/ PeF and DPAG [33]. The anti-panic effects of LY354740 have been studied in panic-prone rats. When pretreated with either the LY354740 or the effective anti-panic drug alprazolam, rats exhibited a dose-dependent inhibition of NaLacinduced panic-like response, without sedative side effects [24]. Therefore, it was thought to be a novel anti-panic drug, without the adverse effects of BDZs. Recently, selective Group II mGlu receptor type 2 (mGluR2) agonists, CBiPES and THIIC, were tested in panic-prone rats. The mGluR2 agonists provide some advantages over the mGluR2/3 agonists because they are activated only under conditions of enhanced glutamate and act only by presynaptic mechanisms. Thus, they selectively reduce excessive synaptic glutamate release without altering the basal glutamate release and with fewer side effects [33]. Pretreating panic-prone rats with CbiPES, THIIC or alprazolam (positive control) blocked NaLac induced paniclike response. These data suggested that mGluR2 agonists may be a promising anti-panic drug [33]. Antagonists of ionotropic NMDA receptors Postynaptic receptor mechanisms of enhanced glutamatergic transmission in panic-prone rats were investigated using receptor antagonists that inhibit the postsynaptic effects of glutamate. NMDA antagonist (MK-801) injection into the DMH/ PeF resulted in a dose-dependent blockade of the NaLacinduced panic-like behavior. Non-NMDA (GYKI52466) 3.1.1.2

injection in the same areas did not prevent panic. Hence, postsynaptic mechanisms involving the NMDA glutamate receptors may contribute to the panic vulnerability [34]. Notwithstanding, human clinical trials on some NMDA antagonists (ketamine, MK-801, CNS-1102) across different disorders reported adverse effects, such as psychotic symptoms [35]. In light of this, the development of compounds targeting the mGluRs appears to be more promising to effectively treat panic [33] and no clinical trials with NMDA antagonists have been conducted in PD. 3.1.2

Clinical studies Group II-metabotropic glutamate receptors agonists

3.1.2.1

A placebo-controlled double-blind randomized multi-centre Phase II study investigated the tolerability and efficacy of LY354740 (Eglumegad) (4 weeks of treatment, 200 mg) in reducing PAs induced by a single inhalation of a 35% CO2/65% O2 gas mixture. Thirty subjects with PD who showed CO2-induced PAs before starting treatment were included. LY354740 was significantly more effective than placebo in reducing CO2-induced PAs after 4 weeks, and the two compounds had similar side effects. Subjects treated with LY354740 reported greater subjective clinical improvement than those receiving placebo [36]. These promising results were not confirmed in another multi-centre placebo-controlled randomized double-blind Phase II efficacy study. Patients with PD received LY354740 (100 or 200 mg), paroxetine (active comparator) (40 mg) or placebo (10 subjects) for 9 weeks. The primary outcome measure was the percentage of subjects without PAs during the final 3 weeks of treatment. Preliminary results on a subsample of 37 subjects have been published [37]. LY354740 was well tolerated but did not differ from placebo in any treatment effect. Of note, in this study, there was a very high response rate in placebo and, unexpectedly, paroxetine, an effective first-line anti-panic treatment, showed an effect only on secondary measures (global anxiety symptoms and illness severity). The study was terminated due to the lack of efficacy of LY354740. Poor oral bioavailability of LY354740 may partly explain these results and, therefore, the pro-drug form LY544344 may be investigated in future studies [38]. Preliminary Phase I studies suggest an efficacy of LY544344 in reducing anxiety responses to cholecystokinin tetrapeptide (CCK-4) infusion in healthy human volunteers [39]. However, since the validity of CCK-4 challenge as experimental model of PAs is still unclear [17], studies with well-validated experimental methods to induce panic are needed to test the anti-panic properties of LY544344. No clinical studies tested the anti-panic efficacy of the selective mGluR2 agonists. Clinical results are scarce and inconclusive; therefore, further clinical trials are worthy of being conducted given the promising preclinical data. Antagonists of orexin receptors The ORX neuropeptide is synthesized in the ORX neurons located exclusively in the DMH/PeF and lateral 3.2


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hypothalamus. The two active forms, ORXA and ORXB, arise from a common prepro-ORX precursor and act via two G-protein-coupled ORX receptors, ORX1R/ORX2R. The ORX system modulates several biological processes, including respiratory and cardiovascular functions [40], and ORX neurons have CO2/H+ chemosensitive properties. Prepro-orexin knockout mice exhibit reduced ventilatory response to inspired CO2 (5 -- 10%). Infusion of an ORX1R antagonist (SB334867) into the rostral medullary raphe of mice produced significant decrease in the ventilatory response to 7% CO2 [40]. Exposing rats to hypercarbic (20% CO2), normoxic gas results in anxiety behavior and cardiorespiratory responses associated with increased cellular responses in the DMH/PeF ORX neurons [41]. In panic-prone rats, the ORX neurons are selectively activated after NaLac infusion and this activation is associated with panic-like responses, while silencing the hypothalamic ORX gene (Hcrt) product blocks the NaLac-induced panic-like responses [23]. Finally, ORX system projects to brain regions implicated in defense and emotional responses, such as the periaqueductal grey and the limbic system [42]. These findings suggest that ORX may be a key substrate implicated in hypersensitive ‘suffocation/chemoception monitor’ and panic-like responses. Accordingly, increased levels of ORX were found in the cerebrospinal fluid (CSF) of subjects with panic anxiety compared to subjects without panic anxiety [23], and chronic treatment with sertraline, an effective anti-panic drug, lowered ORX levels in the CSF, whereas bupropion, an antidepressant with a low efficacy in treating PD, did not [43]. 3.2.1

Preclinical studies Antagonists of ORX1 receptors

3.2.1.1

The anti-panic properties of ORX1R antagonists have been tested in panic-prone rats. Pretreatment with ORX1R antagonists SB334867 or SB408124 attenuated panic-like responses after NaLac infusion, without significant sedative side effects [44]. In line with this, pretreating rats with SB334867 attenuated anxiety behavior and pressor response following exposure to 20% CO2 [41]. The ability of ORX1R antagonists in reducing the hyperreactivity to panicogenic stimuli suggest that they may constitute a novel approach for treating PD. Clinical studies To the best of our knowledge, no clinical studies have been conducted on ORX1R receptors antagonists. 3.2.2

Corticotrophin-releasing factor 1-receptor antagonists

3.3

Corticotrophin-releasing factor (CRF) is a neuropeptide released from the paraventricular nucleus of the hypothalamus that activates the hypothalamo-pituitary-adrenal (HPA) axis, ultimately resulting in glucocorticoids release. In addition to its role in the HPA axis, CRF is synthesized in several brain regions, including those coordinating behavioral/autonomic reactions in defense responses and putatively implicated in PAs 6

generation (e.g., DPAG, DMH) [45]. Two G-protein-linked CRF receptors have been identified, CRF1 (especially in the cortex, limbic system, brainstem, cerebellum) and CRF2 (especially in the hypothalamus) [46]. Polymorphisms of the CRF1 receptor gene have been associated with panic vulnerability in German/ Japanese population samples [47,48]. Infusion of CRF in DPAG facilitates escape behaviors both in the ETM and the electrical stimulation of the DPAG in rats [49]. In the ETM, CRF also facilitates inhibitory avoidance acquisition, suggesting an additional anxiogenic effect beyond the panicogenic one [49]. Finally, CRF1 receptors activate ORX neurons, which are implicated in the pathophysiology of PAs [17].

3.3.1

Preclinical studies CRF 1-receptor antagonists

3.3.1.1

Both in the ETM and the electrical stimulation of the DPAG, pretreatment with the CRF1 receptor antagonist antalarmin blocked the increase of escape behaviors induced by infusion of CRF in DPAG of rats [49], whereas nor the administration of CRF alone into the DMH neither that of CRF combined with antalarmin altered escape response in the ETM [45]. This discrepancy may partly arise from lack of objective physiological measures in the ETM model that may hide potential respiratory/autonomic panic-like responses following DMH stimulation [6]. Accordingly, in panic-prone rats, intraperitoneal pretreatment with a selective, non-peptidergic CRF1 receptor antagonist (JNJ19567470/CRA5626) had an acute effect in preventing the NaLac-induced panic-like behavior and cardiovascular responses, without sedative effects [50]. Although mixed, these data suggest CRF1 receptor antagonists could be a promising anti-panic drug.

3.3.2

Clinical studies CRF1 receptor antagonist R317573

3.3.2.1

A Phase I randomized, double-blind, placebo-controlled study investigated the effects of the non-peptidergic, selective CRF1 receptor antagonist R317573 (40 mg, 7-day treatment) on 7.5% CO2 inhalation (20 min) response in 32 healthy subjects [46]. Treatment with R317573 showed significant efficacy in reducing the CO2-induced behavioral response. The drug was safe, well tolerated, without sedative effects. However, despite the 7.5% CO2 inhalation was previously used as experimental model of PAs [51], it has been recently proposed as potential model of generalized anxiety disorder [46]. Consequently, in this study, ‘arbitrary’ measures of significant response to inhalation were used to investigate general anxiety symptoms rather than PAs. Thus, no conclusions can be drawn about the specific anti-panic properties of the R317573. Well-validated experimental procedure to induce PAs in laboratory, such as the 35%CO2-65%O2 challenge test, and with standardized outcome measures to define challenge-induced PAs are recommended [52].




3.4

Angiotensin II receptor antagonists

Angiotensin II (A-II) mechanisms act on cardiovascular, renal, endocrine and peripheral nervous systems. In addition, A-II is present in several brain areas involved in a wide range of emotional/anxiety processes. Investigation of the role of A-II in the pathophysiology of panic is at an early stage; however, preliminary findings suggest that A-II may be implicated in PAs generation. Neurons producing A-II are present in the CVOs, which detect changes in several plasma parameters (e.g., pH, osmolarity) [53] and whose connections with the DMH/PeF appear critical for NaLac-induced responses in panic-prone rats [26]. Accordingly, direct injections of A-II into the DMH/PeF of panic-prone rats elicited panic-like responses, which were blocked by pretreatment with saralasin an A-II receptor antagonist [22,53]. A-II may modulate respiratory variability, which appeared to be increased in subjects with PD [54,55]. Indeed, when compared to saline injections, intracerebroventricular administration of A-II receptor antagonist saralasin reduced respiratory rate and tidal volume variability in rats [56]. Finally, genetic polymorphisms of the ACE, which catalyses the conversion of angiotensin I to the more potent A-II, have been associated with PD in humans [57,58]. 3.4.1

Preclinical studies A-II receptor antagonists losartan and saralasin

3.4.1.1

Injection of the A-II type 1 receptor antagonist losartan and the non-specific A-II receptor antagonist saralasin into the DMH of panic-prone rats blocked the panic-like behavioral and cardio-respiratory NaLac-induced responses [53]. This suggests a potential usefulness of A-II receptors antagonists in treating PAs. Clinical studies To the best of our knowledge, no clinical studies have been conducted on A-II receptor antagonists. 3.4.2

The modulators of endocannabinoid system The main active component of cannabis sativa, D9-tetrahydrocannabinol (D9-THC), is known to have biphasic effects, thus reducing anxiety or inducing dysphoria/anxiety and PAs in humans [59], depending on the dose and the emotional state prior to use [60]. Similar effects were found with agonists of cannabinoid type-1 (CB1) receptors in several animal models of anxiety, depending on the distinct roles of receptors in different brain regions, the drug and dose range used and the differential sensitivity of the receptors to different compounds [61]. D9-THC exerts its effects through the CB1 receptor, for which the main endogenous ligand is anandamide (AEA). Recent findings showed that the anxiolytic effects of AEA are CB1 receptor-mediated, whereas the anxiogenic effects are mediated by transient receptor potential vanilloid type-1 (TRPV1) channels [62,63]. The endocannabinoid (eCB) system is a ubiquitous signaling system that has important regulatory functions in the CNS. Some findings 3.5

suggested a potential role of this system in PAs. CB1 and TRPV1 receptors and other components of the eCB system are present in brain areas associated with defensive responses, including the DPAG and hypothalamus. CBs modulate the release of several neurotransmitters implicated in panic pathophysiology, such as serotonin, glutamate and GABA [63]. Direct injection of cannabidiol (a non-psychotomimetic component of cannabis sativa) in the DPAG decreased escape responses induced by local electrical stimulation [64], as well as repeated peripheral administration of cannabidiol decreased escape responses in the ETM, probably by facilitating 5HT1A-mediated neurotransmission in the DPAG [65]. Preclinical studies Modulators of eCB system capsazepine and URB597

3.5.1

3.5.1.1

Intra-DPAG administration of the TRPV1 receptors antagonist capsazepine decreased escape responses induced by electrical stimulation of DPAG [66] or by exposure to the ETM test [67], probably by allowing endogenous AEA to act entirely on the CB1 receptors and/or decreasing the TRPV1-mediated glutamate release. The inhibitors of AEA degradation by the enzymes fatty acid amide hydrolase (FAAHs) enhance eCB system activity. In the ETM test, the inhibitor of AEA hydrolysis URB597 decreased escape responses [68]. These eCB-modulating compounds seem to reduce risk of abuse and the biphasic dose-response effects of the CB1 agonists [69]. Therefore, the dual FAAH/TRPV1 blocker N-arachidonoylserotonin, which combines inhibition of AEA degradation and TRPV1 receptor antagonism, may be a promising compound but to date it has been tested only in model of general anxiety, such as EPM [70,71]. Pharmacological manipulation of the eCB system may represent a favorable anti-panic treatment. Preclinical studies in panic-prone rats are recommended. Clinical studies To the best of our knowledge, no clinical studies have been conducted on modulators of the eCB system. 3.5.2

The translocator protein (18 kDa) ligands The translocator protein (18 kDa) (TSPO) transports cholesterol from the outer to the inner mitochondrial membrane. This is the rate-limiting step in the synthesis of pregnenolone, which is the precursor of all other neurosteroids [72]. Ring Areduced neurosteroids are positive allosteric modulators of the GABAA receptors, which occupy a binding site at GABAA receptors different from that of BDZs [73]. Neurosteroids may be implicated in PAs, even though their specific role in the different clinical phenomena of PD is not clear cut. Reduced TSPO expressions were found in the lymphocytes and platelets of subjects with PD with separation anxiety symptoms [74]. Increased levels of allopregnanolone and other neurosteroids were found in subjects with PD, which may reflect counter-regulative mechanisms against the occurrence of PAs and/or to improve high baseline anxiety levels and 3.6


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reduced GABAA receptor sensitivity [75,76]. Women with PD showed weaker 35%CO2-provoked panic responses during the midluteal than during early follicular phases, probably due to progesterone-induced decrease of chemosensitivity to hypercapnia [77]. Experimental panic provocation by NaLac/ CCK-4 challenges decreased allopregnanolone concentration in subjects with PD but not in healthy controls, possibly due to failure in compensatory mechanisms to maintain/ increase neurosteroid levels in response to panicogenic stimuli [78]. Recently, a female-specific association in PD between polymorphisms in genes related to progesterone/ allopregnanolone pathway and anticipatory anxiety levels was found, whereas association with severity of PAs was not found [79].

The neurokinin 1 receptor antagonists Tachykinins are peptide neurotransmitters, including substance P (SP) and neurokinin (NK) A/B, which bind to the transmembrane NK receptors NK1, NK2 and NK3. They are distributed in several brain regions involved in emotional/anxiety states and learning/memory processes [80]. In rats, substantial concentrations of SP were found in the DPAG, a putative critical site for PAs generation [14], while a widespread reduction of brain NK1 receptor binding was found in humans with PD compared to healthy controls [81]. Although very limited, these findings suggested a potential role of SP and NK receptors in PAs/PD [80,82]. 3.7

3.7.1

Preclinical studies The NK1 receptor antagonist spantide

3.7.1.1

3.6.1

Preclinical studies The TSPO (18 kDa) ligand XBD173

3.6.1.1

XBD173 (AC-5216, Emapunil) is a novel selective phenylpurine high-affinity TPSO ligand that indirectly potentiates GABAergic neurotransmission through generation of neurosteroids, with no direct effects at the GABAA receptors sites [72]. Similarly to alprazolam, in a preclinical study acute pretreatment with XBD173 prevented anxiety-like behaviors (SI test) induced by NaLac infusion in panic-prone rats (cardio-respiratory parameters were not assessed). Contrary to alprazolam, no sedation or tolerance was found [73]. Lack of physiological measures in this study does not allow to draw reliable conclusions on the anti-panic effects of XBD173 in this model.

3.6.2

Clinical studies The TSPO (18 kDa) ligand XBD173

3.6.2.1

A Phase I randomized study investigated the effects of 7 consecutive days with placebo, or 10/30/90 mg/day XBD173 or 2 mg/day alprazolam on the responses to CCK-4 challenge in 71 healthy male volunteers. CCK-4-induced anxiety response was significantly reduced after both alprazolam and the highest dose of XBD173. Contrary to alprazolam, XBD173 and placebo showed similar side effects and no withdrawal symptoms [73]. These findings suggest that XBD173 may be a promising fast-acting anxiolytic drug with less severe side effects than BDZs. However, the CCK-4 challenge is not a completely validated human experimental model of PAs [17]. Thus, reliable conclusions on anti-panic properties of XBD173 cannot be drawn. Finally, TPSO are highly expressed in peripheral tissues and TSPO ligands enhance overall neurosteroid synthesis. Therefore, medium- and long-term effects of TSPO ligands as well as their side effects profile under prolonged application need further investigation [73]. Overall, the available data do not allow to understand to what extent modulation of neurosteroids may impact on PAs or on other anxiety symptoms of PD. 8

In rats, NK1 receptor antagonist spantide, but not the NK-3 antagonist SB222200, reduced escape behaviors induced by electrical stimulation of the DPAG, suggesting potential anti-panic properties of NK1 receptor antagonists [82]. Clinical studies The NK1 receptor antagonists vestipitant and vofopitant 3.7.2

3.7.2.1

A Phase I study in 19 healthy subjects pre-screened for their responsiveness to 7% CO2 inhalation (20 min), investigated the efficacy of two NK1 antagonists in blocking the behavioral and physiological (respiratory parameters, heart rate, skin conductance) panic responses to the challenge. The study was randomized, double-blind, placebo-controlled, with alprazolam as active comparator. A single oral dose of vestipitant (GW597599) (15 mg), a single i.v. dose of vofopitant (Gr205171) (25 mg) or a single oral dose of alprazolam (0.75 mg) were administered before the 7% CO2 challenge. Compared with placebo, vestipitant significantly reduced the VAS-Anxiety scale scores, which are the primary measure of a panic-induced response, while alprazolam significantly decreased the panic symptoms list total scores. Vofopitant did not show any panicolytic effect, possibly because of its high pharmacokinetics variability coefficient. No drugs showed effects on the physiological parameters [80]. These findings suggest a potential efficacy of NK1 antagonist vestipitant in preventing behavioral component of PAs. However, to the best of our knowledge, no preclinical studies tested the anti-panic properties of these two compounds using validated animal model of panic, while they showed anxiolytic properties in other models of anxiety and in some clinical studies in humans with social phobia or post-traumatic stress disorder [80]. Overall, theoretical and preclinical bases about the involvement of NK1 receptors in PAs are poorly clarified and other preclinical studies are needed to understand the role of this system in panic. Arginine vasopressin V1B receptor antagonists Arginine vasopressin (AVP) is a cyclic nonapeptide implicated in the activation of the HPA-axis. AVP binds to the V1A, V1B 3.8




and V2 receptors. In particular, the V1B are expressed mainly in the pituitary neurons that secret adrenocorticotropic hormone and in several hypothalamic/limbic regions modulating emotional processes [83]. Some V1B receptor antagonists exhibit antidepressant and anxiolytic effects in several animal models of depression and anxiety [83]. The role of the HPAaxis in PAs is still controversial. Most studies showed no or inconsistent cortisol response in laboratory-induced PAs in humans [84], while activation of the HPA-axis is observable during anticipatory anxiety/anxious behaviors in PD [85]. On the other hand, sustained activation of the HPA-axis may produce increased reactivity to suffocative stimuli in subjects with PD and to increased occurrence of PAs [85]. 3.8.1

Preclinical studies AVP V1B receptor antagonist TASP0233278

3.8.1.1

The anti-panic properties of the V1B receptor antagonist TASP0233278 were studied in panic-prone rats. Pretreatment with TASP0233278, as well as the BDZ chlordiazepoxide (CDP), blocked anxiety-like behavioral response (SI test) to NaLac infusion. Conversely, TASP0233278 did not significantly counteract NaLac-induced respiratory response, while CDP significantly blocked it. This suggests that modulating the HPA axis by V1B receptor antagonists does not play a crucial role in preventing PAs [83]. This might be partly explained by a limited involvement of the HPA-axis in PAs generation.

Clinical studies To the best of our knowledge, no clinical studies involving NPS have been conducted. 3.9.2

Isoxazoline derivative ABIO-08/01 ABIO-08/01 (BTG-1640) is a new isoxazoline derivative currently under development for treating anxiety disorders, including PD. Its mechanism of action is unclear. It seems to influence the metabolism of neurotransmitters throughout the inhibition of the calmodulin-dependent II kinase. During increased neuronal stimulation, ABIO 08/01 seems to increase the ratio between GABA and glutamate, which is implicated in panic pathophysiology [89]. 3.10

Preclinical studies It has been reported that ABIO-08/01 showed anxiolytic/cognition-enhancing effects in several animal models of anxiety, with no addiction/withdrawal or sedative effects [89]. No data are available on these studies. 3.10.1

Clinical studies A Phase II multi-centre, double-blind, randomized, placebocontrolled increasing-dose study has been completed to evaluate clinical effects, safety and tolerability of ABIO 08/01 in patient with PD [90]. No results are available. 3.10.2

4. 3.8.2

To the best of our knowledge, no clinical studies on AVP V1B receptor antagonist have been conducted. Neuropeptide S Neuropeptide S (NPS) regulates various biological functions by the G-protein-coupled NPS receptors (NPSRs) [86]. Preliminary findings suggested NPS is involved in PD susceptibility, even though it is unclear to which extent it may be implicated in PAs generation or in other phenomena following PAs occurrence. Indeed, NPSRs modulate the release of many neurotransmitters involved in panic pathophysiology, such as glutamate and serotonin, but they are mainly expressed in brain areas implicated in conditioned behaviors/anticipatory anxiety, such as the amygdala, hippocampus and medial prefrontal cortex [87]. Polymorphisms of the NPSR gene have been associated with PD in women, but this association seems to be mediated through elevated anxiety sensitivity, autonomic arousal and distorted processing of fear-related stimuli [88]. 3.9

Preclinical studies In the ETM test, NPS given intracerebroventricularly elicited both anxiolytic (reduced inhibitory avoidance) and panicolytic (reduced escape response)-like effects [87]. Further studies are needed to understand to what extent NPS may modulate PAs or other related phenomena, such as conditioned/anxiety behavior. 3.9.1

Conclusion

Clinical studies

In order to improve pharmacotherapy in PD, alternative novel mechanisms-based approaches need to be considered. Disappointingly, in the last years there has been little progress. Glutamate- and ORX-related molecular targets may represent the most promising opportunities for treating PAs. Animal studies supported the implication of these systems in panic vulnerability. Both mGlu II receptor agonists and ORX1R antagonists blocked panic-like responses in a preclinical model that adequately reflect PAs (the panic-prone rats), without the adverse effects of BDZs. Despite scientific evidence encourages glutamate/ORX-related approaches development, clinical validation levels remain unsatisfactory. No human studies with ORX1R antagonists were conducted, and results of the only two studies with mGlu II receptor agonists are inconclusive. Considering the robust theoretical bases and the promising preclinical data about the involvement of these systems in the neurobiology of PAs, further clinical studies are recommended. Investigation of other compounds is at an early stage and only preliminary data are available. However, some of these seem promising and worthy of being investigated in depth. Indeed, antagonists of CRF1 and A-II receptors showed anti-panic properties in panic-prone rats, which is in agreement with the potential role of the CRF/AII systems in the neurobiology of panic. Similarly, modulators of the eCB system may play a role in PAs generation, even though they have been tested only in the ETM and further studies are warranted. Conversely, the available data, albeit


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scarce, suggest that the V1B receptor antagonists are not a promising treatment for PAs, probably due to the limited involvement of these systems in the pathophysiology of PAs. NPS showed both panicolytic and anxiolytic properties in only one study with ETM, and thus other preclinical data are needed before drawing conclusions. Finally, modulation of neurosteroids may play a role in neurobiology of panic, but the available data on TSPO ligands do not allow to clarify to which extent they may be effective on PAs or on other anxiety symptoms of PD. Finally, only one Phase I clinical study suggested one NK1 antagonist may have anti-panic properties; because the theoretical and preclinical bases about the involvement of this system in PAs generation are poorly clarified, other preclinical studies should be performed before implementing further clinical studies. Overall, we conclude that new medications for PD are far away from being implemented. 5.

Expert opinion

Our overview shows that no novel compound for treating PAs is ready for clinical use in subjects with PD. The reasons are multiple. Translational validity of the available animal models is a main limit. Developing models of PAs with high face, construct and predictive validity is challenging [20]. The neurobiology of PAs is not fully understood and probably results from complex interactions between genetic vulnerability and environmental factors [6,14]. This may affect the construct validity of the models currently used. In addition, although both the ETM and the panic-prone rats are considered models in which activation of DPAG occurs [14,27], these paradigms have some substantial differences. In the former, a ‘normal’ animal is placed in a situation of unconditioned threat and the escape behaviors are considered panic-like responses, while, in the latter, animals are vulnerable to NaLac and the NaLac-induced cardio-respiratory responses are the main measures of panic-like responses. This may result in differences in face and construct validity between the two models, thus affecting the interpretation and translation of the results to clinical studies. The models based on behavioral changes in animals without measuring objective physiological responses suffer of higher risk of biases related to inferential assessment of emotionality or behaviors. Therefore, it is more difficult to understand whether novel compounds tested only with these models may be effective on PAs or on other anxiety symptoms of PD. To increase the validity and comparability of the panic models, translational biomarkers should be always included [91]. A biomarker-based approach offers advantages in selecting appropriate targets and outcomes to reduce the heterogeneity in both preclinical and clinical studies [3]. This approach is still at an early stage in PD, although the elements needed for this do exist. Panic has the unique feature among psychopathological phenomena that it can be reliably provoked in the laboratory. The best validated method to date is the administration of CO2. Hypersensitivity 10

to CO2/chemoceptive stimuli is considered a biomarker of vulnerability to PAs in humans [7], and it has been repeatedly shown that, depending on dosage, panic can also be induced in healthy volunteers with that method as well [92,93]. Thus, it may be a good translational biomarker candidate. Together with the recently proposed [94] panic provocation in mice using CO2, this opens the avenue for a truly translational experimental panic model, using the same panic-evoking stimulus in rodents, healthy human volunteers and PD patients. Provocation of unconditioned reactions to NaLac/ hypercapnia and including cardio-respiratory measures could be the ‘gold standard’ to reproduce human PAs, albeit comparable outcome measures in animals and human need to be defined. These models are not extensively used in preclinical studies. NaLac infusion in panic-prone rats tested only part of the novel anti-panic compounds, while animals with heightened respiratory sensitivity to CO2 inhalation and aversion towards CO2-enriched environments [95] have not been used in pharmacological preclinical studies. In addition, the predictive capacity of animal models of panic may be improved by using inbred strains with different genetic background. In humans, treatment response to traditional antipanic drugs is associated to allelic variations of 5-HTTPRL, 5-HT1A receptor and catechol -O-methyltransferase (all of which modulate neurotransmission) [96-98]. Therefore, in animal models, allelic variations of those genes modulating the systems targeted by new anti-panic drugs might be used. Other criticalities appear in human studies. Potential antipanic compounds selected by panic-prone rats were not sufficiently tested or were not tested at all in human trials. Biomarker- and endophenotype-based approaches have been poorly implemented. The NaLac/CO2 challenge-induced PAs have been scarcely used to confirm the anti-panic properties of those compounds selected in preclinical studies and often the induced-responses have been measured with heterogeneous criteria. In addition, other less-validated methods to induce PAs have been used (e.g., CCK-4 challenge), thus affecting the validity of results. Subjects with PD showed respiratory irregularities, impaired autonomic and balance system functions, which may be associated with different clinical profiles [11]. CO2 sensitivity is considered an endophenotype of panic and is associated with respiratory symptoms, higher occurrence of PAs and familiarity for PD, probably characterizing a respiratory subtype (RS) [7]. Notwithstanding, in clinical trials subjects with PD are considered a homogeneous group with homogeneous outcomes. This affects the validity of the results and may explain some discrepancies between promising results of preclinical studies and unsatisfactory ones of clinical studies. Implementing the use of clinical profiles/patterns of neurobiological functions in pharmacological research could help to select truly homogeneous groups of patients and test the efficacy of compounds on specific symptoms and functions. In ‘personalized medicine’, reliable indicators of prediction/outcome are essential for choosing the most appropriate medications for sub-




populations of patients with specific neurobiological/ symptomatological features. Accordingly, ORX may be mainly effective in the RS-panic. As well, anti-muscarinic drugs and opioid receptors agonists could be future tools for RS-panic, because the former decrease sensitivity to hypercapnia in subjects with PD [99], while the opioid system is implicated in hypersensitivity to panicogenic challenges, respiratory abnormalities and vulnerability to PAs [10]. However, these pharmacological options are not under development for PAs. In future, personalized anti-panic treatments may be implemented using artificial neural networks to explore those factors affecting treatment response heterogeneity, including gender, familiarity, clinical profiles, biomarkers and allelic variations modulating treatment responses [96]. The discussed limitations may also partly explain why anti-panic drugs currently used are ineffective in a significant proportion of patients. Indeed, different efficacy across different subgroups of patients has been scarcely investigated [4]. Overall, ‘personalized psychiatry’ may represent a promising alternative for developing new anti-panic medications. This approach is in line with the NIH RDoC (Research Domain Criteria), an ongoing initiative aimed at shifting the focus of research away from existing diagnostic categories and towards observable behavior and neurobiological measures. Research on different domains (e.g., ‘negative valence systems’, including fear/anxiety) is structured around ‘units of analysis’ which include genes, molecules, cells, brain circuits and behaviors [100]. For some psychiatric symptoms, including PAs, Bibliography

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The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

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Affiliation

Giampaolo Perna†1,2,3 MD PhD, Koen Schruers2,4 MD PhD, Alessandra Alciati1 MD & Daniela Caldirola1 MD PhD † Author for correspondence 1 Hermanas Hospitalarias - Villa San Benedetto Menni Hospital, Department of Clinical Neurosciences, FoRiPsi, via Roma 16, 22032, Albese con Cassano, Como, Italy Tel: +39031 4291511; Fax: +39031 427246; E-mail: [email protected] 2 University of Maastricht, Medicine and Life Sciences, Department of Psychiatry and Neuropsychology, Faculty of Health, Maastricht, The Netherlands 3 University of Miami, Leonard Miller School of Medicine, Department of Psychiatry and Behavioral Sciences, Miami, FL, USA 4 Faculty of Psychology, University of Leuven, Center for Learning and Experimental Psychology, Loeven, Belgium


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