FI128750B - Methods for determining the therapeutic efficacy of rapid-acting antidepressants and personalized antidepressant therapy related thereto - Google Patents

Abstract

The present invention relates to the fields of life sciences, medicine, monitoring, therapies and drug screening and development. Specifically, the invention relates to a method for determining the therapeutic efficacy of a rapid-acting antidepressant, a real-time method of optimizing antidepressant treatment and a method of screening novel rapid-acting antidepressants and/or plasticity enhancers. Still, the present invention relates to a method of treating a subject with a rapid-acting antidepressant.

Description

Methods for determining the therapeutic efficacy of rapid-acting antidepres- sants and personalized antidepressant therapy related thereto

FIELD OF THE INVENTION

The present invention relates to the fields of life sciences, medicine, monitoring, therapies and drug screening and development. Specifically, the invention relates to a method for determining the therapeutic efficacy of a rapid-acting antidepressant, the present disclosure relates to a real-time method of optimizing antidepressant treatment and the present invention relates to a method of screening novel rapid- acting antidepressants and/or plasticity enhancers. Still, the present disclosure re- lates to a method of treating a subject with a rapid-acting antidepressant.

BACKGROUND OF THE INVENTION

Major depression is a highly disabling psychiatric condition, the most significant risk factor for suicide and one of the biggest contributors to the disease burden world- wide. Depressive disorders produce immeasurable human suffering and enormous economic burden. Depressed mood, anhedonia, lack of concentration, feelings of worthlessness and suicidal thoughts are common symptoms of depression. Yet, conventional antidepressants alleviate these symptoms very slowly, if at all. Indeed, many patients don’t respond to prescription antidepressants, and in those who do the therapeutic effects become evident with a considerable delay.

The huge unmet medical need for better antidepressants is evidenced by the ongo- ing medical use of electroconvulsive therapy (ECT). An electric current leading into

S a short epileptic-like of EEG (electroencephalogram) activity is delivered in ECT un-

N der light anesthesia, but how this seizure leads into a remedy remains poorly under- e stood. The therapeutic effects of ECT emerge faster than those of conventional an- n 30 — tidepressants, yet rapid reduction of depressive symptoms already after a single

I ECT treatment is only seldom reported. a i Rapid antidepressant effects of subanesthetic ketamine has been well established

R in clinical trials and the treatment is already in off-label use in various countries, > 35 including the USA. Reported response rates to ketamine are somewhat impressive, but significant amounts of patients remain treatment-refractory (Aan Het Rot et al. 2012, Biol. Psychiatry 72, 537-547). To this end, extensive research input has been put forward to find predictive efficacy markers and to uncover the precise neurobio- logical basis underlying the rapid antidepressant effects of ketamine. Although cat- egorized as a non-competitive NMDA (N-methyl-D-aspartate) receptor antagonist, ketamine has rich pharmacology and regulates several other targets as well includ- ing the AMPA (a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid), the opioid and the cholinergic receptors, several ion channels and enzymes. Intriguingly, a re- cent animal report suggests that it is the metabolic byproduct of ketamine, a putative positive AMPA receptor modulator cis-6-hydroxynorketamine (HNK), which is solely responsible for the rapid antidepressant effects of ketamine (Zanos P et al. 2016,

Nature 533, 481-486). Despite promising animal studies, potential rapid antidepres- sant effects of positive AMPA receptor allosteric modulators have not been thor- oughly assessed in clinical trials.

Rapid antidepressant effects of N>O (nitrous oxide or laughing gas), another NMDA receptor blocker, have also been studied. Nagele et al have published a research article (Nagele et al, 2015, Biol. Psychiatry vol. 78, pages 10-18) and have filed a patent application (publication WO2015/175531 A1) regarding the use of N>O (5- 75%) as a sole agent or in combination with other specific drugs and treatments for the treatment of depressive disorders. The neurobiological mechanism underlying the therapeutic effects of N2O remain, however, obscure.

On the other hand, systems and methods for use in characterization and discovery of neuroactive drugs have also been published. E.g. WO2016/029211 A1 describes a system for evaluating an effectiveness of one or more drugs administered to a subject. Said system utilizes analyzes of the neurophysiological data to generate signatures indicative of brain states induced by one or more drugs administered to

S the subject. Furthermore, US 2012/0165696 A1 describes a method for assessing

N the susceptibility of ahuman individual suffering from a psychiatric condition or neu- & rological disorder to neuromodulation treatment, wherein said method comprises n 30 the use of electroencephalographic (EEG) dataset. There is, however, no biomoni-

I tor, whether based on neurophysiological measure or biological readout, in the clin- = ical domain that would reliably predict or optimize rapid antidepressant effects. s

R Furthermore, US2008159958 A1 relates to a method for evaluating a test compound > 35 wherein a histamine-1 receptor (H1R) antagonist is administered to an animal to produce a change in the recorded brain wave potentials and administering a hista-

mine-3 receptor (H3R) antagonist in the same animal to determine a dose that de- creases the effects of the H1R antagonist on EEG. US2013295016 A1 concerns disease-relevant biomarkers and more specifically it relates to methods for deter- mining the presence or absence of a signature in electroencephalographic oscilla- tions recorded from a subject. US2010016751 A1 relates to a method for identifying subjects at risk for adverse effects from a psychotropic or CNS-active treatment.

US2008021345 A1 relates to a method of indicating a subject's reaction to different agents administered to induce anesthesia.

There is a need to better understand rapid antidepressant mechanisms and to find predictive biomarkers to control their efficacy. There also remains a significant un- met need for more effective and safer therapies alleviating the symptoms of depres- sive disorders.

BRIEF DESCRIPTION OF THE INVENTION

One object of the present invention is to provide methods and tools for systematic testing of the antidepressant effects in clinical and preclinical settings. Another ob- ject of the present invention is to provide tools and a method for effective and spe- cific treatment of depressive disorders. The objects of the invention are achieved by utilizing a surprising and a simple biomarker for planning and optimizing personal- ized antidepressant therapy. Defects of the prior art, including but not limited to in- effective rapid-acting antidepressant treatments and lack of straightforward, reliable and safe monitoring methods in the living brain, are thus overcome by the present invention.

S It has now been found that it is possible to predict the individual therapeutic re-

N sponses to a rapid-acting antidepressant by analyzing time-lapsed changes in spe- & cific neurophysiological markers in real-time after administering said treatment. The n 30 results of the monitoring enable prediction of the therapeutic efficacy and optionally

I outcome of the rapid-acting antidepressant treatment in said subject. The present = invention thus enables real time monitoring combined with optimized treatment in i any subject. As an example, a subject who does not benefit from a standard rapid-

R acting antidepressant treatment or its specific dosing regimen may be found quickly > 35 after administration of said antidepressant and thus the treatment may be modified for optimal outcome or replaced for another treatment very early. In particular, the present invention discloses a method allowing to rapidly modify and adjust the ther- apeutic effects of a rapid-acting antidepressant and to reproduce evoked brain re- sponses beneficial against depression in remarkable precision and timescale.

Therefore, the present invention provides a very effective and personal antidepres- sant efficacy biomonitoring tool.

The results of a study presented in this disclosure encourage systematic testing of time-lapsed slow neural oscillations as reliable efficacy monitors of the antidepres- sant effects in clinical settings. The present invention is based on the idea of provid- ing a method, wherein slow neural oscillations from the cortex of the brain are mon- itored by electrophysiological monitoring (e.g. using the EEG), in real-time, in a spe- cific embodiment in a non-invasive way. Indeed, the present invention solves the problems of conventional unsuccessful, slow and unspecific therapies. The present invention surprisingly reveals predictive efficacy markers to be utilized in antidepres- sant treatment, e.g. optimizing an antidepressant treatment. The present invention may also be utilized for development of novel rapid-acting antidepressants e.g. in clinical and preclinical settings.

There is currently no method in the clinical domain that would reliably predict rapid antidepressant efficacy or any specific method to optimally titrate the dosing of rapid antidepressant in each patient. The present invention overcomes said deficiencies.

Moreover, intermittent dosing consisting of repeated exposures to rapid-acting anti- depressants with short intervals during the same treatment session are enabled by the present invention.

Indeed, it has now been surprisingly found that an interplay between “excitation” (E)

S and “inhibition” (I) in the cortex of the brain can be utilized for determining the ther-

N apeutic efficacy of rapid-acting antidepressants. The present invention enables cou- & pling of cortical excitability and resulting rebound slow neural oscillations for study- n 30 ing the effects of rapid-acting antidepressants. The results of the present disclosure

I show that transient regulation of cortical excitability and emerged slow neural oscil- = lations evoked by such excitability is a shared neurobiological phenomenon for treat- i ments that can bring immediate amelioration of depressive symptoms (see e.g. Fig-

R ures 13 and 14). Remarkably, such homeostatic transient alterations in E-I balance > 35 can be rapidly reproduced with specific pharmacological and/or non-pharmacologi- cal means.

The present invention enables reproducible and efficient production of rapid antide- pressant effects. By the present invention it is possible to produce and rapidly re- produce brain states, which are beneficial against depression or other nervous sys- tem disorders associated with compromised plasticity (as used herein brain plastic- 5 ity refers to lasting change of the brain). Actually, the present invention is based on methods and means to reliably and effectively control and monitor produced excita- tory and inhibitory responses in the brain and optionally to rapidly and repeatedly reproduce said responses.

Further, the present disclosure shows association between ongoing regulation of

TrkB and GSK3B signaling pathways and slow neural oscillations. Also, a surge of glutamatergic neuronal excitability and synthesis of plasticity-related activity-de- pendent immediate early genes (e.g. Arc, Bdnf) is pointed out as a shared neurobi- ological feature for rapid-acting antidepressants.

In short, effects of rapid antidepressants can be studied and optimized by utilizing the present invention and furthermore by aid of the present invention it is possible to develop novel more efficient treatments against depression and other treatments wherein antidepressants and induced plasticity is considered beneficial.

The present invention relates to a method for determining a therapeutic efficacy of a rapid-acting antidepressant, wherein the method comprises: monitoring slow neural oscillations from the cortex of the brain of a subject administered with one or more rapid-acting antidepressant(s) by electrophysiologi- cal monitoring, and determining a therapeutic efficacy of said rapid-acting antidepressant(s) based

S on fluctuations (e.g. dynamic fluctuations) on slow neural oscillations before the ad-

N ministration and during E phase (excitation) and | phase (inhibition) after the admin- & istration under the influence of said rapid-acting antidepressant(s) in said subject. n 30

I Also, the present disclosure relates to a real-time method of optimizing antidepres- = sant treatment, wherein the method comprises i monitoring slow neural oscillations from the cortex of the brain of a subject

R administered with one or more rapid-acting antidepressant(s) by electrophysiologi- > 35 cal monitoring, and determining a therapeutic efficacy of said rapid-acting antidepressant(s) based on fluctuations (e.g. dynamic fluctuations) on slow neural oscillations before the ad- ministration and during E phase (excitation) and | phase (inhibition) after the admin- istration under the influence of said rapid-acting antidepressant(s) in said subject, and optimizing the rapid-acting antidepressant treatment.

Still, the present invention relates to a method of screening novel rapid-acting anti- depressants, wherein the method comprises monitoring slow neural oscillations from the cortex of the brain of a subject administered with a pharmaceutical by electrophysiological monitoring, and determining a rapid-acting antidepressant therapeutic efficacy of said pharma- ceutical based on fluctuations (e.g. dynamic fluctuations) on slow neural oscillations before the administration and during E phase (excitation) and | phase (inhibition) after the administration under the influence of said pharmaceutical and/or other in- — tervention in said subject.

Still further, the present disclosure relates to a method of treating a subject with a rapid-acting antidepressant, wherein the method comprises: monitoring slow neural oscillations from the cortex of the brain of a subject to be administered with one or more rapid-acting antidepressant(s) by electrophysio- logical monitoring, administering to the subject in need thereof one or more rapid-acting antide- pressant(s), monitoring slow neural oscillations from the cortex of the brain of the subject administered with one or more rapid-acting antidepressant(s) by electrophysiologi- cal monitoring, and

S determining a therapeutic efficacy of said rapid-acting antidepressant(s) based

N on fluctuations (e.g. dynamic fluctuations) on slow neural oscillations before the ad- & ministration and during E phase (excitation) and I phase (inhibition) after the admin- n 30 istration under the influence of said rapid-acting antidepressant(s) in said subject. = - Still further, the present disclosure relates to a rapid-acting antidepressant for use i in treating a subject having a nervous system disorder associated with compromised

R plasticity, wherein > 35 slow neural oscillations are monitored from the cortex of the brain of a subject to be administered with one or more rapid-acting antidepressant(s) by electrophys- iological monitoring,

one or more rapid-acting antidepressant(s) are to be administered to the sub- ject in need thereof, slow neural oscillations are monitored from the cortex of the brain of the subject administered with one or more rapid-acting antidepressant(s) by electrophysiologi- cal monitoring, and a therapeutic efficacy of said rapid-acting antidepressant(s) is determined based on fluctuations (e.g. dynamic fluctuations) on slow neural oscillations before the administration and during E phase (excitation) and | phase (inhibition) after the administration under the influence of said rapid-acting antidepressant(s) in said sub- ject.

And still further, the present disclosure relates to a method for determining concur- rent TrkB activation and GSKB inhibition (e.g. indirectly), wherein the method com- prises monitoring slow neural oscillations from the cortex of the brain of a subject by electrophysiological monitoring, wherein optionally antidepressant-induced TrkB activation and GSK inhibition is indirectly determined when the electrophysiological monitoring reveals more slow oscillations in the | phase compared to the E phase. Furthermore, optionally it is possible to determine TrkB and GSKB signaling from the brain tissue using molec- ular biology methods (e.g. assaying the kinase activity or posttranslational modifica- tion that alter the activity state of given protein).

And still furthermore, the present disclosure relates to a method for determining — concurrent TrkB activation and GSK inhibition (e.g. indirectly) by monitoring a sed- ative state of said subject.

S

N Other objects, details and advantages of the present invention will become apparent & from the following drawings, detailed description and examples. n 30

I BRIEF DESCRIPTION OF THE DRAWINGS a i Figure 1 reveals that rapid-acting antidepressants facilitate cortical excitability that

R evokes a transient rebound emergence of slow EEG oscillations during which TrkB > 35 and GSK3B signaling becomes regulated. (A) Biological markers implicated in ac- tivity-dependent neuronal firing and antidepressant effects (c-fos, arc, bdnf, zif-268, homer-1A, egr-2, mkp-1 and synapsin mRNA) are up-regulated 1-hour after 60 min

N20 (50%) treatment while phosphorylation of TrkBY86 (indicate increased activity),

GSK3B?? (indicate reduced activity) and p70S6k1421/424 (indicate increased activity) remain unaltered. (B) Biological markers implicated in activity-dependent neuronal firing (p-MAPK"T202Y204 and c-fos mRNA) are up-regulated during N2O (50%) admin- istration while phosphorylation of TrkBY816, GSK3[3%® and p70S6k1421/424 remain un- altered. (C) c-fos, arc and bdnf mRNAs levels are up-regulated to the same magni- tude by 2-hour continuous N20 (50%) and 1-hour N2O (50%) followed by an hour washout period. (D) Representative time frequency EEG spectrogram and power of major EEG oscillations immediately before, during and after N2O (50%) administra- tion. Slow-wave EEG oscillations (delta, theta) transiently emerge upon gas with- drawal. (E) Rebound slow-wave delta EEG oscillations after discontinuation of 75%

NO treatment. (F) Phosphorylation levels of TrkBY816, GSK3BS9 and p70S6k1421/424 are increased at 5-minute post-N>O exposure (50-75%). (G) Subanesthetic dose of ketamine (10 mg/kg, i.p.) evokes rebound slow-wave delta EEG oscillations gradu- ally and only after the drug-induced high gamma oscillations have subsided. (H)

Flurothyl-induced seizures evokes rebound emergence of slow-wave EEG oscilla- tions. Levels of p-TrkBY8'6, p-GSK3RS® and p70S6k1421/424 are increased at 15 min after withdrawal of flurothyl. Data are means + S.E.M. *<0.05, **<0.01, ***<0.005.

Figure 2 reveals that sedative-anesthetic doses of ketamine regulate TrkB and

GSK3B signaling while subanesthetic ketamine and cis-6-hydroxynorketamine has negligible acute effects on these molecular events. (A) Phosphorylation of TrkBY816,

GSK3BS9 and p70S6k1421/424 in the adult mouse medial prefrontal cortex 30 min after an i.p. injection of saline (SAL), cis-6-hydroxynorketamine (HNK, 20 mg/kg) or ket- amine (KET, 10 mg/kg, 100 mg/kg). (B) Effects of KET and 6,6-dideuteroketamine (d-KET, 100 mg/kg, i.p.; 30min) on p-TrkBY8'®, p-GSK3BS9 and p-p70S6k1421/424. (C)

S Representative time freguency EEG spectrograms immediately before and during

N HNK and KET treatment. (D) Power of major EEG oscillations during HNK and KET & treatment. Sedative-anesthetic dose of KET increase most EEG oscillations includ- n 30 ing slow-wave delta and theta and gamma oscillations, while subanesthetic KET

I and HNK produce more subtle effects. (E) Amphetamine, a pharmacological stimu- - lant, produces no acute effects on TrkB and GSK3B signaling. Data are means + i S.E.M. *<0.05, **<0.01, ***<0.005.

R

> 35 Figure 3 further confirms the dose-dependent effects of ketamine on TrkB and

GSK3B signaling. (A) Phosphorylation of TrkBY816, GSK3BS9 and p70S6k 21/424 in the mouse medial prefrontal cortex 30-minutes after an acute i.p. injection of keta- mine (10 mg/kg, 50 mg/kg, 200 mg/kg; i.p.). (B) Phosphorylation of TrkB"8i6,

GSK3BS9 and p70S6k 21424 in the adult mouse medial prefrontal cortex 3-minutes after an acute i.p. injection of high dose of ketamine (200 mg/kg; i.p.). Data are means + S.E.M. *<0.05, **<0.01.

Figure 4 further reveals the dose-dependent acute effects of ketamine on slow EEG oscillations. Power of major EEG oscillations during 30-minute ketamine (1, 7.5, 10, 50 mg/kg, i.p.) treatment. Data are means + S.E.M.

Figure 5 reveals the effects of intermittent (i.e. repeated) nitrous oxide (N20, 75%) treatment on EEG. (A) Power of beta, gamma, theta and alpha oscillations in male mice before, during and after NO. Note the emergence of slow-wave theta EEG oscillations upon gas withdrawal. (B) Power of major EEG oscillations in female mice before, during and after NO treatment. Note the emergence of slow-wave delta and theta EEG oscillations upon gas withdrawal. Data are means + S.E.M.

Figure 6 reveals increased phosphorylation of TrkB, GSK3B and p70S6k after with- drawal from 65% NO. Phosphorylation of TrkBY816, GSK3BS9 and p70S6k 21/424 in the mouse medial prefrontal cortex at 15-minutes after discontinuing N2O (65%, 20 min). Data are means + S.E.M. *<0.05, **<0.01.

Figure 7 reveals gradual rebound emergence of slow EEG oscillations (1-4 Hz) after subanesthetic dose of ketamine (7.5 mg/kg, i.p.) in female mice. Note that slow EEG oscillations emerge after the acute effects of ketamine on (high) gamma oscillations have subsided.

S

N Figure 8 reveals that direct facilitation of slow-wave EEG oscillations (delta, theta) and “antidepressant-like” phosphorylation responses in TrkB and GSK3B under the n 30 influence of hypnotic-sedative drug medetomidine is not translated into behavioral

I changes associated with antidepressant responses. (A) Representative time fre- - guency EEG spectrograms and power of major EEG oscillations during 30-minute i saline and medetomidine (0.3 mg/kg, i.p.) treatment. (B) A low dose of medetomi-

R dine (0.05 mg/kg, i.p.) rapidly increases phosphorylation of TrkBY8!6, GSK3BS? and > 35 —p708S6k"42142%4 while reduces phosphorylation of MAPKT202/1204 (indicates reduced glutamatergic firing), in the mouse medial prefrontal cortex. (C) Dose-dependent effects of ketamine on phospho-MAPKT™202¥204 (D) Number of escape failures be- fore and 24 hours after subanesthetic ketamine (15 mg/kg, i.p.) or medetomidine (0.05 mg/kg, i.p.) in the learned helplessness paradigm. Data are means + S.E.M. *<0.05, **<0.01, ***<0.005.

Figure 9 reveals the acute effects of hypnotic-sedative drug gaboxadol (THIP) on

EEG and TrkB signaling. (A) Phosphorylation of TrkBY816 and p70S6k™21424 in the adult mouse medial prefrontal cortex 30 min after an acute i.p. injection of gaboxadol (10 mg/kg; i.p.) or saline (SAL). (B) Power of major EEG oscillations during 30 min gaboxadol treatment. Data are means + S.E.M. *<0.05, **<0.01.

Figure 10 reveals the effects of tricyclic drug imipramine (an antidepressant that alleviates depression very slowly) slow EEG oscillations and GSK3B phosphoryla- tion. (A) Phosphorylation of TrkBY816 p70S6k1421/424 and GSK3B$? in the adult mouse medial prefrontal cortex 30 min after an acute i.p. injection of imipramine (50 mg/kg; i.p.) or saline (SAL). (B) Power of major EEG oscillations during 30 min gaboxadol treatment. Data are means + S.E.M. **<0.01, ****<0.001.

Figure 11 reveals the acute effects of medetomidine on immediate early gene ex- pression. Levels of c-fos, arc, bdnf, homeria and zif-268 mRNA in the adult mouse medial prefrontal cortex remain unaltered 2 hours after an acute i.p. injection of me- detomidine (0.3 mg/kg; i.p.) or saline. Data are means + S.E.M.

Figure 12 reveals the acute effects of medetomidine on EEG. Power of major EEG oscillations during 30 min medetomidine treatment. Data are means + S.E.M.

S Figure 13 shows the essential time-lapsed interplay between “excitation” (E phase)

N and “inhibition” (I phase) caused by rapid-acting antidepressants. Rapid-acting an- & tidepressants produce cortical excitability that evokes a homeostatic emergence of n 30 slow neural oscillations, during which molecular events intimately implicated with

I rapid antidepressant effects become altered: activation of TrkB receptor and inhibi- - tion of GSK3B (glycogen synthase kinase 3B). Such evoked homeostatic brain re- i sponses beneficial against depression can be rapidly produced and reproduced and

R controlled with interventions capable of producing transient cortical excitability. Mon- > 35 itoring of the time-lapsed emergence of slow wave neuronal network oscillations before and during the treatment(s) can be utilized to control and monitor antidepres- sant efficacy.

Figure 14 illustrates three example schemes (in a time line) enabled by the present invention. Scheme 1 describes a method for determining a therapeutic efficacy of a rapid acting antidepressant by monitoring slow neural oscillations. Desired altera- tions of slow neural oscillations reveal the presence of therapeutic effects (e.g. re- bound oscillations or more slow neural oscillations in the | phase compared to the E phase). Schemes 2 and 3 describe a real-time method for optimizing rapid acting antidepressant treatment by monitoring slow neural oscillations. If a desired re- sponse is not achieved with a rapid acting antidepressant treatment said treatment may be e.g. repeated or modified or replaced with another rapid acting antidepres- sant in order to arrive at a desired or improved outcome (e.g. more slow neural oscillations in the | phase compared to the E phase). Schemes 1-3 are also appli- cable e.g. for methods of screening novel rapid acting antidepressants or combina- tions thereof.

DETAILED DESCRIPTION OF THE INVENTION

One object of the present invention is to provide a method for determining the ther- apeutic efficacy of a rapid-acting antidepressant. As used herein “rapid-acting anti- depressant” refers to is a type of antidepressant which improves symptoms of de- pression quickly, within minutes to hours. Rapid-acting antidepressants are a dis- tinct group of antidepressants compared to conventional antidepressants, which re- quire weeks of administration for their therapeutic (e.g. antidepressant) effects to manifest. In one embodiment of the invention the rapid-acting antidepressant is a pharmacological compound that has one or more of the following properties: NMDA-

R blockade (e.g. NMDA-R antagonists, ketamine, N2O) and/or GABAaA-R blockade

S (e.g. GABA4-R antagonists, flurothyl) and/or GABAa-R positive allosteric modulation

N (e.g. gamma-hydroxybutyrate) and/or GHB-R agonism (gamma-hydroxybutyrate, 3- e hydroxycyclopent-1-enecarboxylic acid (HOCPCA)) and/or AMPA-R positive allo- n 30 steric modulation (e.g. positive allosteric modulators of the AMPA-R, hydroxynorket-

I amine) and/or 5-HT2A-R agonism (e.g. psilocybin) and/or alfa2-R antagonism (e.g. = atipamezole) and/or antimuscarinic (e.g. scopolamine) and/or up-regulate immedi- i ate-early genes and/or produce seizures and/or evoke glutamate release; or any

R related pharmaceutical or any combination thereof. In one embodiment of the inven- > 35 tion the rapid-acting antidepressant is a pharmacological compound selected from the group consisting of: NMDA-R antagonist (e.g. NMDA-R antagonists, ketamine,

N20), GABAA-R antagonist (e.g. GABA4-R antagonists, flurothyl), GABAa-R positive allosteric modulator (e.g. gamma-hydroxybutyrate), GHB-R agonist (gamma-hy- droxybutyrate, 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA)), AMPA-R pos- itive allosteric modulator (e.g. positive allosteric modulators of the AMPA-R, hy- droxynorketamine), 5-HT2A-R agonist (e.g. psilocybin), alfa2-R antagonist (e.g. atipamezole), and antimuscarinic (e.g. scopolamine), and any combination thereof.

In one embodiment of the invention the rapid-acting antidepressant may be any pharmaceutical regulating excitation (i.e. E phase) with favorable kinetics (e.g. half- life (t12): 1 s — 4 hours). In a further embodiment the rapid-acting antidepressant(s) is(are) a non-pharmacological antidepressant selected from the group consisting of sleep deprivation, electroconvulsive therapy (ECT), (repetitive) transcranial mag- netic stimulation (TMS), transcranial direct current stimulation (tDCS), vagal nerve stimulation, photic stimulation, direct current stimulation, hyperthermia, hypother- mia, cortical cooling, or any related non-pharmacological method, or any combina- tion thereof.

Rapid-acting antidepressants of one type may be utilized in the present invention but alternatively two or more different types of rapid-acting antidepressants may be combined for the method of the present invention. In one specific embodiment the rapid-acting antidepressants are combined with other pharmaceuticals (e.g. one or more rapid-acting or conventional antidepressants, or any other pharmaceutical(s)) or non-pharmaceutical treatments. In a specific embodiment the rapid-acting anti- depressants are a combination of one or more pharmacological rapid-acting antide- pressants and one or more non-pharmacological rapid-acting antidepressants (e.g. selected from the groups of pharmacological and non-pharmacological rapid-acting antidepressants listed in the preceding paragraph).

S In the present invention slow neural oscillations are monitored from the cortex of the

N brain of a subject administered with one or more rapid-acting antidepressant(s) by & electrophysiological monitoring. Neural oscillation is rhythmic or repetitive neural n 30 activity in the nervous system. Oscillatory activity can be driven either by mecha-

I nisms within individual neurons or by interactions between neurons. Synchronized = activity of large numbers of neurons can give rise to macroscopic oscillations, which i can be observed by electrophysiological monitoring including but not limited to elec-

R troencephalogram (EEG) and/or magnetoencephalography (MEG). The interaction > 35 between neurons can give rise to oscillations at a different frequency than the firing frequency of individual neurons. Oscillatory activity may respond to pharmaceuticals or non-pharmaceutical treatments e.g. by increases or decreases in frequency and/or amplitude, or show a temporary interruption. Neurons may change the fre- quency at which they oscillate. In one embodiment of the invention “slow neural oscillations” refer to oscillations that have their frequency range between 1 — 6 Hz (delta, low theta).

In the present invention slow neural oscillations are monitored from the cortex of the brain. “The cortex of the brain” refers to the cerebral cortex, the most anterior brain region comprising an outer zone of neural tissue called gray matter, which contains neuronal cell bodies.

As used herein “electrophysiological monitoring” refers to any monitoring of the presence, absence, amount or changes of any electrophysiological character (e.g. slow neural oscillations) of a subject or any part thereof, e.g. in vivo, ex vivo or in vitro. In one embodiment of the invention the electrophysiological monitoring is EEG and/or MEG and/or other mean. EEG is an electrophysiological monitoring method to record electrical activity of the brain. EEG is typically a noninvasive method, wherein the electrodes are placed along the scalp, but invasive EEG (intracranial

EEG, IEEG) may also be utilized for the present invention. EEG measures voltage fluctuations resulting from ionic current within the neurons of the brain. The type of neural oscillations can be observed in EEG signals in the frequency domain. Mag- netoencephalography (MEG) is a functional neuroimaging technique for mapping brain activity by recording magnetic fields produced by electrical currents occurring naturally in the brain, using very sensitive magnetometers.

Brain thermo- and energy regulations are implicated in antidepressant effects and generation of slow neural oscillations. Brain oscillatory rhythms are also regulated

S in a circadian manner and through homeostatic control mechanisms. Notably, slow-

N wave delta oscillations (0.5-4 Hz) are characteristic features of non-REM deep & sleep, sedation and drowsiness. n 30

I The therapeutic efficacy of a rapid-acting antidepressant(s) utilized in the present = invention is determined based on temporal fluctuations on slow neural oscillations i before the administration and during E phase (excitation) and | phase (inhibition)

R after the administration under the influence of said rapid-acting antidepressant(s) in > 35 a subject. Most importantly, the ability of the treatment to generate sufficient but transient “E phase” determines the rebound emergence of “| phase”. That said, a treatment that directly regulates “I phase” without preceding “E phase” is not con- sidered therapeutic. The "I phase” can be readily monitored by quantifying slow neu- ral oscillations. Moreover, the emergence of rebound slow neural oscillations indi- rectly monitor also the preceding “E phase”. Slow neural oscillations remain unal- tered or may reduce during “E phase” compared to the period of prior applying any treatment (e.g. baseline). “E phase” may also be monitored by motor evoked poten- tials and the ability of any putative rapid-acting antidepressant to have properties required to generate sufficient “E phase” can be predetermined in preclinical exper- iments by investigating its effects on markers implicated in cortical excitation. For some specific treatments (e.g. ketamine), transient emergence of high gamma os- cillations indicates ongoing “E phase”.

In one embodiment of the invention differences of slow neural oscillations before and after administration of a rapid-acting antidepressant (e.g. decreased or no slow neural oscillations during E phase and increased slow neural oscillations during phase; increased slow neural oscillations during E phase and decreased or no slow neural oscillations during | phase), are used for determining the therapeutic efficacy or predicting the outcome of the therapy in a subject. In a specific embodiment dif- ferences of slow neural oscillations before administration of a rapid-acting antide- pressant and after administration of said rapid-acting antidepressant during E phase (e.g. decreased or no slow neural oscillations compared to slow neural oscillations before administration) and during | phase (e.g. increased slow neural oscillations compared to E-phase) are used for determining the therapeutic efficacy or predicting the outcome of the therapy in a subject.

In one embodiment of the invention the electrophysiological monitoring revealing

S more slow oscillations in the | phase compared to the E phase indicates the thera-

N peutic efficacy or good outcome of the rapid-acting antidepressant. On the other & hand in one embodiment the electrophysiological monitoring revealing less slow n 30 neural oscillations in the | phase compared to the E phase, or no slow oscillations

I in the I phase, or no slow oscillations in the I and E phases, indicates lack of thera- = peutic efficacy, poor therapeutic efficacy or poor outcome of the rapid-acting antide- i pressant. As used herein “more slow neural oscillations” refers to more slow neural

R oscillations measured by cumulative amount of high-amplitude slow neural oscilla- > 35 tions. As used herein “less slow oscillations” refers to less slow oscillations meas- ured by cumulative amount of high-amplitude slow neural oscillations. In a very spe- cific embodiment the electrophysiological monitoring revealing at least 5%, 10%,

15%, or more (e.g. at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%) slow oscillations in the | phase compared to the E phase indicates the therapeutic efficacy or outcome of the rapid-acting anti- depressant. In the present invention the presence and/or absence and/or amount of slow neural oscillations may be used for indicating the therapeutic efficacy of the rapid-acting antidepressant.

The specific properties and effects of a given rapid-acting antidepressant treatment determines the duration of the “E phase”. As for some specific treatments the “E phase” may last only 1-30 seconds (e.g. flurothyl, ECT), although more sustained (1 min — 120 min) “E phase” may be considered safer and more efficient (e.g. keta- mine, nitrous oxide). In one embodiment of the invention the duration of the E phase is 1 second — 2 hours. In one embodiment of the invention the duration of the | phase is 5 min — 1 hour. In a further embodiment of the invention the duration of the com- bination of E and | phases is 5 min — 3 hours. In one embodiment of the invention duration of the E phase is about 30 seconds that produces rebound emergence of "I phase” lasting about 10-30 min. In another embodiment of the invention the dura- tion of the E phase is about 5 seconds that produces rebound emergence of “I phase” lasting about 5-10 min.

In a very specific embodiment of the invention concurrent (i.e. simultaneous) emer- gence of E phase and | phase indicates therapeutic efficacy or good outcome of the therapy of the rapid-acting antidepressant(s) in a subject.

In some embodiments of the present invention, the slow neural oscillations or wave- forms thereof or lack of slow neural oscillations in the EEG segment representing

S the period when a subject is under the influence of a rapid-acting antidepressant are

N compared to reference slow neural oscillations or waveforms thereof or lack of slow & neural oscillations. A reference waveform is the waveform in the EEG segment be- n 30 fore administration of the rapid-acting antidepressant. In a very specific embodiment

I it is possible to classify slow neural oscillations or waveforms (e.g. as either slow = waves or not slow waves) via numerical outputs, which are generated based on slow i neural oscillations or waveforms of a subject under the influence of a rapid-acting

R antidepressant and a reference waveform. > 35

As used herein “under the influence of a rapid-acting antidepressant(s)” refers to a time-period when a rapid-acting antidepressant has direct pharmacological or phys- iological effects on a subject. Said time-period varies depending on the rapid-acting antidepressant(s) (e.g. t12) and may be selected e.g. from prior art publications or based on the common general knowledge of a skilled artisan. Examples of suitable periods include but are not limited to e.g. about 1 second - 3 hours for nitrous oxide and about 5 min - 120 min for ketamine.

As used herein “a therapeutic efficacy” refers to an ability to ameliorate any harmful effects of the nervous system disorder associated with compromised plasticity, such as including but not limited to depression, sleepiness, sleep problems, feeling anx- ious, mood swings, psychosis, hallucinations, weight gain, suicidal thoughts, disturb- ing thoughts, feelings or dreams, mental or physical distress to trauma-related cues, attempts to avoid trauma-related cues, alterations in how a person thinks and feels, neurodegeneration, addiction and brain trauma. In one embodiment the therapeutic efficacy is for nervous system disorder associated with compromised plasticity, e.g. a disorder is selected from the group consisting of depression, anxiety, addiction, neurodegenerative disorder, brain trauma, post-traumatic stress disorder, and neu- ropathic pain. As used herein depression refers to any type of depression e.g. major depression, chronic depression (dysthymia), atypical depression, postpartum depres- sion, bipolar depression (manic depression), seasonal depression (SAD), psychotic depression and/or treatment-resistant depression. Anxiety or anxiety disorders are a group of mental disorders characterized by feelings of anxiety and fear. Neuro- degenerative disorders are a group of conditions which primarily affect the neurons in the human brain. When neurons of the nervous system (including the brain and spinal cord) become damaged or die they cannot be replaced by the body. Exam-

S ples of neurodegenerative diseases include Parkinson's, Alzheimer’s, and Hunting-

N ton's disease. Neuropathic pain is pain caused by a damage or disease affecting & the somatosensory nervous system. n 30

I A therapeutic effect of administration of a rapid acting antidepressant may be as- = sessed by monitoring the slow neural oscillations and/or any other characteristics i e.g. symptoms of a subject such as selected from the group consisting of, but not

R limited to, depression, sleepiness, sleep problems, feeling anxious, mood swings, > 35 psychosis, hallucinations, weight gain, suicidal thoughts, disturbing thoughts, feelings or dreams, mental or physical distress to trauma-related cues, attempts to avoid trauma-related cues, alterations in how a person thinks and feels, neurodegenera- tion, addiction and brain trauma.

Therapeutically effective amount of a rapid acting antidepressant refers to an amount with which the harmful effects of a nervous system disorder associated with compromised plasticity, e.g. depression, anxiety, post-traumatic stress disorder, neurodegenerative disorder, neuropathic pain, or addiction, are, at a minimum, ame- liorated. The effects of rapid acting antidepressants may be either short term or long term effects. “Treatment” or “treating” refers to administration of a rapid acting antidepressant for purposes which include not only complete cure but also prophylaxis, amelioration, or alleviation of disorders or symptoms related to nervous system disorder associ- ated with compromised plasticity, e.g. a disorder is selected from the group consist- ing of depression, anxiety, post-traumatic stress disorder, neurodegenerative disor- der, neuropathic pain, and addiction. In one embodiment of the disclosure the rapid- acting antidepressant is or in one embodiment of the invention the rapid-acting an- tidepressant has been administered intravenously, intra-arterially, intramuscularly, intranasally, by an oral administration or by inhalation. Any conventional method may be used for administration. A person skilled in the art knows how and when to administer a rapid-acting antidepressant, depending e.g. on the type of the rapid- acting antidepressant and formulation thereof as well as a subject and symptoms or disease of said subject. In one specific embodiment a rapid-acting antidepressant is a pharmaceutical composition comprising at least a therapeutically effective agent. In addition, a pharmaceutical composition may also comprise any other ther- apeutically effective agents, any other agents, such as a pharmaceutically accepta-

S ble solvent, diluent, carrier, buffer, excipient, adjuvant, antiseptic, filling, stabilizing

N or thickening agent, and/or any components normally found in corresponding prod- & ucts. The pharmaceutical composition may be in any form, such as in a solid, sem- n 30 — isolid or liquid form, suitable for administration. A formulation can be selected from

I a group consisting of, but not limited to, solutions, emulsions, suspensions, spray, = tablets, pellets and capsules. The pharmaceutical compositions may be produced i by any conventional processes known in the art.

R

> 35 In a specific embodiment a rapid-acting antidepressant is administered or has been administered on the same day when the therapeutic efficacy is determined. In one embodiment monitoring of the slow neural oscillations or determination of the ther- apeutic efficacy is repeated once or twice or several times after the subject has further been administered with the rapid-acting antidepressant for the second time, third time or several times e.g. during the same day as the first administration, re- spectively, or after the subject has further been administered with another rapid- acting antidepressant.

Additionally, the administration of a rapid-acting antidepressant can be combined to the administration of other therapeutic agents. The administration can be simulta- neous, separate or sequential. The administration of a rapid-acting antidepressant can also be combined to other forms of therapy, such as psychotherapy, and may be more effective than either one alone. In one embodiment of the invention a rapid- acting antidepressant is utilized as the only therapeutically active agent.

In a very specific embodiment a therapeutic state of the brain is obtained by the method of the present invention, wherein the cortex of the brain of a subject admin- istered with a rapid acting antidepressant is monitored. A therapeutic state of the brain refers to a state, which causes, enables or augments therapeutic effects e.g. amelioration of the symptoms of a subject. On the other hand, a therapeutic state of the brain may also refer to a therapeutically optimal state of the brain e.g. for psy- chotherapy to take its best effects. In such a case administration of a rapid-acting antidepressant does not necessarily cause amelioration of the symptoms in a sub- ject by itself but enables optimal effects of rehabilitation. Indeed, the present inven- tion enables personalized treatment of a subject, e.g. when combined with any non- pharmaceutical therapy such as psychotherapy.

S In one embodiment the method of the present invention further comprises monitor-

N ing neurophysiological data, behavioral data, respiratory data, blood flow data, car- & diac data, galvanic skin response data, data on biochemical marker(s) (e.g. markers n 30 from the blood, serum, urine, brain) or any combination thereof, specifically after the

I administration under the influence of said rapid-acting antidepressant(s) in said sub- = ject. In a further embodiment the behavioral data is selected from the group consist- i ing of data of a guestionnaire study, data of the Hamilton rating scale for depression,

R data of the beck depression inventory, and data of the suicide behaviors guestion- > 35 naire. In a specific embodiment no further monitoring is needed, i.e. e.g. the method comprises monitoring slow neural oscillations from the cortex of the brain of a sub-

ject administered with one or more rapid-acting antidepressant(s) by electrophysio- logical monitoring and no further monitoring is needed, or the method comprises monitoring slow neural oscillations from the cortex of the brain of a subject adminis- tered with one or more rapid-acting antidepressant(s) by electrophysiological moni- toring and further comprises monitoring neurophysiological data, behavioral data, respiratory data, blood flow data, cardiac data, galvanic skin response data, data on biochemical marker(s) or any combination thereof and no further monitoring is needed.

Surprisingly the present invention enables determining TrkB and GSKB signaling alterations indirectly. As used herein determining TrkB and GSKB signaling refers to determining the presence, absence and/or amount of signaling. Indeed, the re- sults of the present disclosure are able to reveal an association between TrkB and

GSKB signaling and slow neural oscillations, e.g. more slow EEG oscillations pre- dicts on-going TrkB activation and GSK3B inhibition in the brain. As used herein “indirectly” refers to a situation wherein TrkB and GSK signaling are indirectly de- termined by monitoring slow neural oscillations, potentiated by the found association between TrkB and GSKB signaling and slow neural oscillations. In a very specific embodiment TrkB and GSKB signaling are indirectly determined by monitoring slow neural oscillations or the sedative state of the subject. As used herein “a sedative state” refers to a state of a subject with reduced irritability or excitement and said sedative state can be monitored e.g. using specific scales. Examples of such scales, which can also be used in the present invention include MSAT (Minnesota Sedation

Assessment Tool), UMSS (University of Michigan Sedation Scale), the Ramsay

Scale (Ramsay, et al. 1974) and/or the RASS (Richmond Agitation-Sedation Scale).

The continuum of sedation may be defined e.g. as follows (American society of An-

S esthesiologists): minimal sedation (normal response to verbal stimuli), moderate se-

N dation or conscious sedation (purposeful response to verbal/tactile stimulation), & deep sedation (purposeful response to repeated or painful stimulation), general an- n 30 — esthesia (unarousable even with painful stimulus). In some context deep sedation

I may also be considered as a part of the spectrum of general anesthesia. a i In the present study (see examples section of the disclosure) it was found that N2O,

R a NMDA-R antagonist and a rapid-acting antidepressant, produce rebound (i.e. after > 35 drug withdrawal) slow EEG oscillations in succession to the facilitation of cortical excitability during gas administration. Most importantly, ongoing slow EEG oscilla- tions co-associate with increased activation of TrkB and inhibition of GSK3B. The intriguing positive correlation between these molecular events coupled with rapid antidepressant effects and slow EEG oscillations — neural oscillations characteristic for deep sleep — was further confirmed with hypnotic-sedative agents. Most im- portantly, the present study further demonstrates that TrkB activation or GSK3 inhi- — bition per se is insufficient in producing antidepressant effects. Instead consecutive regulation of cortical excitability and regulation of TrkB and GSK3B during the re- bound slow EEG oscillations is shared neurobiological phenomenon for interven- tions that can bring rapid antidepressant responses in humans. In particular, the ability of a drug or non-pharmacological procedure to directly augment slow neural oscillations, without the preceding cortical excitability and under the direct influence of said manipulation, does not determine its antidepressant effects.

More specifically, the data of the present disclosure demonstrate that slow neural oscillations — readily and safely captured by the EEG — predict ongoing TrkB activa- — tion and GSK3B inhibition in the brain. First, ketamine dose-dependently regulates activation TrkB (tyrosine phosphorylation / autophosphorylation) and GSK3B inhibi- tion (phosphorylation into the inhibitory serine-9 residue); most prominent effects are evident at doses producing anesthesia and prominent slow neural oscillations.

Notably, subanesthetic, rather than sedative-anesthetic, doses of ketamine are commonly considered as doses relevant with antidepressant effects. Second, hyp- notic-sedative agents that specifically increase slow neural EEG readily recapitulate the effects of ketamine (sedative-anesthetic doses) on TrkB and GSK3B signaling.

The ability of classical antidepressants, such as tricyclic antidepressants, to acutely regulate TrkB and GSK3B signaling is also associated with the emergence of slow wave EEG. Most convincingly, TrkB and GSK3B signaling remain unaltered during

N2O administration when slow neural activity is slightly reduced. Phosphorylation of

S TrkB and GSK3B emerge gradually only after discontinuation of N2O and this is di-

N rectly associated with a rebound increase in slow EEG oscillations. Interestingly, & whereas N>O readily increases activity-dependent immediate early genes it's ef- n 30 fects on slow oscillations (and TrkB and GSK3B signaling) emerge as a response in

I the brain upon discontinuation of the gas flow. As used herein TrkB i.e. tropomyosin = receptor kinase B (also called as neurotrophic receptor tyrosine kinase 2, NTRK2) i refers to the high affinity catalytic receptor for "neurotrophins", which are small pro-

R tein growth factors that induce the survival, maintenance, differentiation of distinct > 35 neuronal populations. Some neurotrophins, in particular BDNF (brain-derived neu- rotrophic factor), also importantly regulates neuronal and synaptic plasticity. Several pharmaceuticals activate TrkB receptors (e.g. antidepressants) and thereby pro- mote neuronal plasticity and provide neuroprotection. The neurotrophins that acti- vate TrkB are BDNF (Brain Derived Neurotrophic Factor), neurotrophin-4 (NT-4), and neurotrophin-3 (NT-3). Human TrkB has e.g. Ensembl accession number

ENSG00000148053 and mouse TrkB has e.g. Ensembl accession number

ENSMUSG00000055254. Tyrosine phosphorylation of TrkB (into tyrosine Y515,

Y705/6 and Y816) can be used as indirect measures of TrkB activity.

As used herein GSK3B is a beta isoform of a glycogen synthase kinase-3 (GSK-3), — which is a proline-directed serine threonine kinase that was initially identified as a phosphorylating and an inactivating agent of glycogen synthase. GSK3B is involved in energy metabolism, neuronal cell development, and body pattern formation.

GSK3B has an EC number EC 2.7.11.1 ((protein-serine/threonine kinase) inhibitor that interferes with the action of tau-protein kinase inhibitor (EC 2.7.11.26)). Phos- — phorylation of GSK3B into the serine-9 residue is associated with reduced GSK3B activity. Inhibition of GSK3B kinase activity is implicated into the therapeutic effects of several distinct pharmaceuticals (e.g. antimanic lithium, rapid-acting antidepres- sant ketamine).

Increased glutamatergic signaling and cortical excitability are strongly connected with the immediate central actions of the most efficient and rapid-acting antidepres- sant therapies, as experimentally evidenced by the activation of mitogen-activated protein kinase (MAPK) and increased expression of activity-dependent immediate early genes (IEGs; e.g. c-fos, arc, bdnf) (de Bartolomeis et al, 2013 Prog. Neuro- —psychopharmacol. Biol. Psychiatry 46, 1-12; Cirelli et al, 1995, J. Sleep Res. 4, 92— 106; Hansen et al, 2007, Cell. Mol. Neurobiol. 27, 585-594; Larsen et al, 2005, Brain

S Res. 1064, 161-165; Li et al, 2010, Science 329, 959-964; Nibuya et al, 1995, J.

N Neurosci. Off. J. Soc. Neurosci. 15, 7539-7547; Taishi et al, 2001, Am. J. Physiol. e Regul. Integr. Comp. Physiol. 281, R839-845). n 30

I As used herein Arc refers to a gene encoding the activity regulated cytoskeleton = associated protein (e.g. Ensembl accession numbers ENSG00000198576 (human) i and ENSMUSG00000022602 (mouse)). Arc is a member of the immediate early

R gene (IEG) family, a rapidly activated class of genes functionally defined by their > 35 ability to be transcribed in the presence of protein synthesis inhibitors. Arc is widely considered to be an important protein in neurobiology because of its activity regula- tion, localization, and utility as a marker for plastic changes in the brain.

As used herein Bdnf refers to a gene encoding brain derived neurotrophic factor (BDNF) (e.g. Ensembl accession numbers ENSG00000176697 (human) and

ENSMUSG00000048482 (mouse)). BDNF acts on certain neurons of the central nervous system and the peripheral nervous system, helping to support the survival of existing neurons, and encourage the growth and differentiation of new neurons and synapses.

In one very specific embodiment of the invention determination of the therapeutic efficacy of one or more rapid-acting antidepressant is carried out in real-time. Real time methods enable efficient, user friendly and safe personalized therapies as well as opportunities to optimize the treatment or dosing of rapid acting antidepressants quickly. In a specific embodiment of the invention monitoring of slow neural oscilla- tions is carried out continuously during the treatment session e.g. before the treat- ment, immediately after administration of a rapid acting antidepressant, during the influence of said rapid acting antidepressant and after the acute pharmacological effects of said rapid acting antidepressant has subsided. As used herein "continu- ously" refers to following up changes of the slow neural oscillations in a non-stop way. Expression "continuously" is opposite to monitoring every now and then or during a specific period of time. In another specific embodiment of the invention monitoring of slow neural oscillations is carried out one or several times (i.e. non- continuously), e.g. during the E phase and | phase such as during specific periods of time of the E phase and | phase.

One object of the present disclosure is to provide a real-time method of optimizing antidepressant treatment. In one embodiment of the disclosure optimizing the rapid-

S acting antidepressant treatment is selected from the group consisting of i) continuing

N said treatment, ii) optimizing the dosing of said rapid-acting antidepressant or the & dosing of another rapid-acting antidepressant, iii) stopping the treatment and iv) n 30 combining said rapid-acting antidepressant treatment with another pharmaceutical

I such as another rapid-acting antidepressant. It is well known to a person skilled in = the art that when a rapid-acting antidepressant has a desired effect, at least based i on the results of monitoring slow neural oscillations, said treatment may be contin-

R ued. However, when a rapid-acting antidepressant does not have a desired effect > 35 at least based on the slow neural oscillations, dosing of said rapid-acting antide- pressant or the dosing of another rapid-acting antidepressant may be optimized for obtaining a desired effect. Alternatively, e.g. when a subject does not respond to a rapid-acting antidepressant said treatment may be stopped. A person skilled in the art also knows when a desired effect could be obtained e.g. by combining the rapid- acting antidepressant treatment with another pharmaceutical such as another rapid- acting antidepressant. Real time monitoring and optimizing methods enable several types of optimizations within a reasonable period of time.

The effective dose of a rapid-acting antidepressant depends on at least the rapid- acting antidepressant in question, the subject in need of the treatment, the type of disease e.g. type of depression, and the level of the disease (e.g. depression). For intravenous ketamine, the dose may vary for example from about 0.4 mg/kg/h to about 1 mg/kg/h, specifically from about 0.4 mg/kg/h to about 0.8 mg/kg/h, and more specifically from about 0.5 mg/kg/h to about 0.7 mg/kg/h. For intranasal ketamine, the dose may vary for example about 25-150 mg (fixed dose). For N>O the dose may vary for example from about 10% to about 75%, specifically from about 30% to about 75%. Under specific conditions, short intermittent exposure(s) of up to 100%

N20 and 100% oxygen may be used. Pharmacokinetically fast rapid-acting antide- pressant, such as N2O, may be administered for example from 1 to 20 times during the same treatment session. Same dosing principles may be applied for concomitant treatment with ketamine and N>O. A desired dosage can be administered in one or more doses at suitable intervals to obtain the desired results. Only one administra- tion of a rapid acting antidepressant may have a therapeutic effect, but specific em- bodiments of the invention require several administrations (e.g. 2-30) during the whole treatment period. The period between administrations may depend on e.g. the patient and type of a disease. In one embodiment of the invention there is a time period of one minute to 24 hours, specifically 2 to 10 hours, between consecutive administrations of rapid acting antidepressants.

S

N In a further embodiment the brain state obtained by administering a rapid-acting & antidepressant is reproduced or optimized for inducing plasticity. n 30

I The present invention may further be utilized for screening novel rapid-acting anti- = depressants or screening an optimal subject for a rapid-acting antidepressant treat- i ment, wherein therapeutic efficacy of a pharmaceutical comprising a rapid-acting

R antidepressant may be determined at least based on fluctuations on slow neural > 35 oscillations before the administration and during E phase (excitation) and | phase (inhibition) after the administration under the influence of said pharmaceutical in said subject. Screening of novel rapid-acting antidepressants in vivo may be carried out by any conventional method known in the art, e.g. in a way wherein a putative rapid- acting antidepressant is administered to a subject (e.g. an animal or a human) one or several times and the therapeutic efficacy is determined as defined in the inde- pendent claims. Selecting an optimal subject or subjects for a rapid-acting antide- —pressant treatment enables personalized and effective treatments. In other words, subjects who most likely benefit from a specific antidepressant treatment will have a normal treatment period, whereas subjects who most likely do not benefit from a specific treatment will receive another kind of treatment or a combination of treat- ments.

Treatment methods are also within the scope of the present disclosure, and then one or more rapid-acting antidepressants are administered to a subject in need thereof. In one embodiment of the disclosure the method of treating a subject with a rapid-acting antidepressant further comprises optimizing the rapid-acting antide- — pressant treatment. “Optimizing the rapid-acting antidepressant treatment” may re- fer to any action, which results in a better therapeutic effect, e.g. including but not limited to changing a dosing of an antidepressant, type of administration, the number of administrations, the antidepressant and a combination of pharmaceuticals. In one embodiment of the disclosure the method of treating a subject with a rapid-acting antidepressant comprises optimizing the rapid-acting antidepressant treatment, wherein optimizing the rapid-acting antidepressant treatment is selected from the group consisting of i) continuing said treatment, ii) optimizing the dosing of said rapid-acting antidepressant or the dosing of another rapid-acting antidepressant, iii) stopping the treatment and iv) combining said rapid-acting antidepressant treatment — with another pharmaceutical such as another rapid-acting antidepressant.

S Before screening an optimal subject or classifying a subject as suitable for the ther-

N apy of the present disclosure or method for determining the therapeutic efficacy of & the present invention, the clinician may for example study any symptoms or assay n 30 any disease markers of the subject. Based on the results deviating from the normal,

I the clinician may suggest a rapid-acting antidepressant treatment of the present in- = vention for the subject. s

R In one embodiment of the invention a subject is a human or an animal, a child, an > 35 adolescent or an adult. In one embodiment a subject is in a need of a treatment or administration of said rapid-acting antidepressant.

Systems and means configured to detect or monitor slow neural oscillations in real time and/or near real time and to be used in the methods of the present invention are also within the scope of the present invention.

It will be obvious to a person skilled in the art that, as the technology advances, the inventive concept can be implemented in various ways. The invention and its em- bodiments are not limited to the examples described below but may vary within the scope of the claims.

EXAMPLES

Materials and methods

Animals

Adult male and female C57BL/6JRccHsd mice (Harlan Laboratories, Venray, Neth- erland) were used. Animals were maintained in the animal facility of University of

Helsinki, Finland, under standard conditions (21°C, 12-hour light-dark cycle) with free access to food and water. The experiments were carried out according to the guidelines of the Society for Neuroscience and were approved by the County Ad- ministrative Board of Southern Finland (License: ESAVI/10527/04.10.07/2014).

Pharmacological treatments

Medical grade N2O (Livopan 50% N20/02 mix, Linde Healthcare; Niontix 100% N2O,

Linde Healthcare). Medical grade oxygen (Conoxia 100% O, Linde Healthcare) was mixed with 100% N20 to achieve >50 (-80%) NO concentrations. Gas was admin- istered into airtight Plexiglass chambers (14 cm x 25 cm x 9 cm) with a flow rate of

S 4-8 |/min. Oxygen or room air was administered for sham animals.

O

N

& To induce myoclonic seizures, 10% flurothyl liquid (in 95% ethanol; Sigma-Aldrich) n 30 were administered into the cotton pad placed inside the lid of an airtight Plexiglass

I chamber (13 cm x 13 cm x 13 cm) at the flow rate of 100 ul/min until the mice ex- - hibited seizures. The lid was removed to terminate the seizure. Animals were eu- i thanized at indicated times (10-60 min) post-seizure.

O

= The following other drugs (and doses) were used: ketamine-HCI, 6,6-d2-ketamine-

N 35 HCI, medetomidine-HCI, dextroamphetamine-HCI, cis-6-hydroxynorketamine-HCI, imipramine-HCI, gaboxadol-HCI. These drugs were diluted in isotonic saline solution and injected i.p. with a final injection volume of 10 ml/kg.

Western blotting and quantitative RT-PCR

Animals were sacrificed at indicated times after the treatments by rapid cervical dis- location followed by decapitation. Bilateral medial prefrontal cortex (including pre- limbic and infralimbic cortices) was rapidly dissected on a cooled dish and stored at -80°C (Antila et al, 2017, Sci. Rep. 7, 7811; Rantamäki et al, 2007, Neuropsycho- pharmacol. Off. Publ. Am. Coll. Neuropsychopharmacol. 32, 2152-2162).

For western blotting the brain samples were homogenized in lysis buffer (137 mM

NaCl, 20 mM Tris, 1% NP-40, 10% glycerol, 48 mM NaF, HO, Complete inhibitor mix (Roche), PhosStop (Roche)) (Rantamäki et al, 2007, Neuropsychopharmacol.

Off. Publ. Am. Coll. Neuropsychopharmacol. 32, 2152-2162). After ~15 min incu- bation on ice, samples were centrifuged (16000g, 15 min, +4°C) and the resulting supernatant collected for further analysis. Sample protein concentrations were measured using Bio-Rad DC protein assay (Bio-Rad Laboratories, Hercules, CA).

Samples (40-50 ug protein) were separated with SDS-PAGE under reducing and denaturing conditions and blotted to a PVDF (polyvinylidene difluoride) membrane (300 mA, 1 hour, 4°C). Membranes were incubated with the following primary anti- bodies (see (Antila et al, 2017)): anti-p-TrkB (#4168; 1:1000; Cell signaling technol- ogy (CST)), anti-TrkB (1:1000; #4603, CST), anti-Trk (sc-11; 1:1000; Santa Cruz

Biotechnology (SCB); ), anti-p-CREB (#9191S; 1:1000; CST), anti-p-p70S6K (#9204S; 1:1000; CST), anti-p-GSK33S® (#9336; 1:1000; CST), anti-p-p44/42-MAP-

KThr202/¥204 (#9106, 1:1000, CST), anti-GSK3B (#9315, 1:1000, CST), anti-p70S6K (#2708, 1:1000, CST) anti-p44/42-MAPK (#9102, 1:1000, CST) and anti-GAPDH (#2118, 1:10 000, CST). Further, the membranes were washed with TBS/0.1%

Tween (TBST) and incubated with horseradish peroxidase conjugated secondary

S antibodies (1:10000 in non-fat dry milk, 1 h at room temperature; Bio-Rad). After

N subseguent washes, secondary antibodies were visualized using enhanced chemi- e luminescence (ECL Plus, ThermoScientific, Vantaa, Finland) for detection by Biorad n 30 ChemiDoc MP camera (Bio-Rad Laboratories, Helsinki, Finland). = - For gPCR, total RNA of the sample was extracted using Trizol (Thermo Scientific) i according to the manufacturer's instructions and treated with DNAse | mix. mRNA

R was reverse transcribed using oligo (dT) primer and SuperScript III Reverse Tran- > 35 — scriptase mix (Thermo Scientific). The amount of cDNA was quantified using real- time PCR. The primers used to amplify specific cDNA regions of the transcripts are shown in Table 1. DNA amplification reactions were run in triplicate in the presence of Maxima SYBRGreen gPCR mix (Thermo Scientific). Second derivate values from each sample were obtained using the LightCycler 480 software (Roche). Relative quantification of template was performed as described previously using standard curve method, with cDNA data being normalized to the control Gapdh and R-actin level

Table 1. Primers used for quantitative RT-PCR.

C (SEQ ID NO: 1) (SEO ID NO: 2)

G (SEO ID NO: 3) (SEO ID NO: 4) (exon IV) | ATAGTAAG (SEO ID NO: 5) | TGAA (SEQ ID NO: 6) tal) GACAAA (SEO ID NO: 7) ATG (SEQ ID NO: 8)

A (SEO ID NO: 9) (SEQ ID NO: 10)

ATC (SEQ ID NO: 11) (SEQ ID NO: 12)

CGG (SEQ ID NO: 13) GG (SEQ ID NO: 14)

CTGG (SEQ ID NO: 15) CC (SEO ID NO: 16)

G (SEO ID NO: 17) (SEQ ID NO: 18)

N A (SEO ID NO: 19) TG (SEO ID NO: 20)

N (egr-1) G (SEQ ID NO: 21) (SEQ ID NO: 22)

E

N EEG recordings and data analysis = 10 For the implantation of electrodes, mice were anesthetized with isoflurane (3% in- = duction, 1.5-2% maintenance). Lidocaine (10 mg/ml) was used as local anesthetic

N and buprenorphine (0.1 mg/kg, s.c.) for postoperative care. Two epidural screw EEG (electroencephalogram) electrodes were placed above the fronto-parietal cortex. A further screw served as mounting support. Two silver wire electrodes were im- planted in the nuchal muscles to monitor the EMG (electromyogram). After the sur- gery, mice were single-housed in Plexiglas boxes. After a recovery period of 5-7 days, animals were connected to flexible counterbalanced cables for EEG/EMG re- cording and habituated to recording cables for three days.

Baseline EEG (10-15 min) recordings of awake animals were conducted prior the treatments. All injection treatments were conducted in the animal's home cages dur- ing light period. N2O treatment was delivered in homemade anesthesia boxes for indicated time periods with a flow rate of 8 I/min.

The EEG and EMG signals were amplified (gain 5 or 10 K) and filtered (high pass: 0.3 Hz; low pass 100 Hz; notch filter) with a 16-channel AC amplifier (A-M System, model 3500), sampled at 254 Hz or 70 Hz with 1401 unit (CED), and recorded using — Spike2 (version 8.07, Cambridge Electronic Devices). The processing of the EEG data was obtained using Spike2 (version 8.07, Cambridge Electronic Devices). EEG power spectra were calculated within the 1-50 Hz frequency range by fast Fourier transform (FFT = 256, Hanning window, 1.0 Hz resolution). Oscillation power in each bandwidth (delta=1—4 Hz; theta=4-7 Hz; alpha=7-12 Hz; beta=12—25 Hz; gamma low=25-40 Hz; gamma high=60-100 Hz) was computed in 30-300-sec epochs from spectrograms (FFT size: 1024 points) for each animal. Representative sonograms were computed using a Hanning window with a block size of 512.

Learned helplessness test

Animals were placed in a shuttle box (Panlab LE100-26, LE900; Software: Bioseb

Packwin) and let habituate for 3 min. On day 1, a pre-test was conducted consisting

S of 140 randomly-paced (at 25, 30 or 35 s intervals) inescapable foot shocks

N (0.45mA, 20 s duration). The pre-test was repeated on day 2. On day 3, testing was & conducted starting with 1 minute habituation and followed by 15 randomly-paced (at n 30 25, 30 or 35 s intervals) escapable shocks (0,45 mA, 20 s duration). During testing,

I animals were able to interrupt the shock delivery/escape by crossing to another = chamber. If the animal failed to escape during the first 10 seconds of a test shock, i the trial was considered as a failure. If more than 50 % of the 15 trials led to a failure,

R the animal was considered helpless. After testing, animals were injected (i.p.) with > 35 saline, ketamine (15 mg/kg) or medetomidine (0.05 mg/kg). Learned helplessness was re-evaluated 24 h post-injection.

Screening of novel rapid acting antidepressants

Any rapid acting antidepressant e.g. medical grade nitrous oxide (N20) or subanes- thetic ketamine is utilized as a positive control and hypnotic-sedative drug (e.g. me- detomidine) utilized as a negative control when novel medicaments are screened for therapeutic effects of rapid acting antidepressants in experimental animals (e.g. rodents). Test medicaments may be prescreened in in vitro settings for their ability to regulate glutamatergic excitation (e.g. immediate early gene expression, phos- phorylation of MAPK) and neural oscillations. Animals, pharmacological treatments,

EEG recordings and data analysis are carried out as described above. All medica- ments having slow neural oscillation profiles resembling those of positive control are forwarded to further studies.

Statistical analyses

Depending on whether data were normally distributed or not, either parametric or nonparametric tests were used for statistical evaluation. In case of more than two groups, analysis of variance (ANOVA) was used. All statistical analyses were per- formed with the Prism 7 software from GraphPad (La Jolla, CA, USA). All tests were used two-sided; a P < 0.05 was considered significant.

Human studies

Human studies with rapid-acting antidepressants are carried out to confirm the re- sults found in animal studies and to correlate EEG observations to clinical outcome (e.g. amelioration of depressive symptoms). After clinical assessment (e.g. symp- toms), an EEG recording set-up will be installed for the subjects. EEG will be rec- orded for 5-30 min at baseline after which the medicaments will be delivered to the subjects during continuous EEG recording. For N2O (50-65%) the EEG will be rec-

S orded during the gas flow (E phase) and upon gas withdrawal (I phase); and the

N treatments repeated for at least once during the same treatment session. For keta- e mine, the EEG will be recorded for 1-4 hours to estimate time-lapsed alterations in n 30 EEG oscillations during the initial phase (E phase) and after the acute pharmaco-

I logical effects have subsided (I phase). Clinical outcome of the treatments may be = assessed at varying time-points post-treatments and correlated retrospectively to i EEG analyses.

R

> 35 Results

Rapid-acting antidepressants facilitate cortical excitability that evokes a tran- sient rebound emergence of slow EEG oscillations during which TrkB and

GSK3B signaling becomes regulated

The remarkable ability of ketamine, a dissociative anesthetic and a drug of abuse, to rapidly ameliorate depressive symptoms after only a single subanesthetic dose has stimulated great enthusiasm among scientists and clinicians (Aan Het Rot et al. 2012, Biol. Psychiatry. 72, 537-547; Berman et al. 2000, Biol. Psychiatry. 47, 351— 354). Reported response rates to ketamine are somewhat impressive, but many patients remain treatment-refractory (Aan Het Rot et al. 2012, Biol. Psychiatry. 72, 537-547). To this end, extensive research input has been put forward to uncover predictive efficacy markers and to nail down the precise pharmacological basis gov- erning the antidepressant effects of ketamine. Although categorized as a non-com- petitive NMDA-R (N-methyl-D-aspartate receptor) blocker, ketamine has rich phar- —macology and it regulates a myriad number of targets. Among them the AMPA-R (a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor) has received con- siderable attention. Emerging evidence suggests that ketamine facilitates glutama- tergic excitability leading into enhanced AMPA-R signaling, which in turn augments synaptic plasticity through the BDNF (brain-derived neurotrophic factor) receptor

TrkB (Autry et al. 2011, Nature. 475, 91-95; Duman and Aghajanian 2012, Science. 338, 68-72; Li et al. 2010, Science. 329, 959-964; Rantamäki and Yalcin, 2016,

Prog. Neuropsychopharmacol. Biol. Psychiatry. 64, 285-292). Inhibition of GSK3B (glycogen synthase kinase 33) kinase, another molecular event associated with ket- amine's therapeutic effects (Beurel et al. 2011, Mol. Psychiatry. 16, 1068-1070), also contributes to enhanced AMPA-R function (Beurel et al. 2016, Bipolar Disord. 18, 473-480). Most notably, a recent preclinical report indicates that the metabolic

S byproduct of ketamine, a putative positive allosteric AMPA-R modulator cis-6-hy-

N droxynorketamine (HNK), is responsible for the antidepressant effects of ketamine & (Zanos et al. 2016, Nature. 533, 481-486). This hypothesis, however, conflicts with n 30 investigations pinpointing the critical role of NMDA-R blockade and the promising

I clinical observations with some other NMDA-R antagonists (Collingridge et al. 2017, - Biol. Psychiatry. 81, e65-e67). Of these agents the nitrous oxide (Nagele et al. i 2015, Biol. Psychiatry. 78, 10-18) (N20, “laughing gas”) is particularly interesting

R since it has very fast kinetics and is essentially un-metabolized in the body. Specif- > 35 ically, the brain concentrations of N2O and thereby its direct pharmacological actions rapidly cease upon gas discontinuation; well before the antidepressant effects be- come evident.

To understand rapid antidepressant mechanisms of NO, we adopted the treatment protocol used in the clinical study by Nagele et al in depressed patients (Nagele et al. 2015, Biol. Psychiatry. 78, 10-18) and investigated how N2O regulates biological markers implicated in rapid antidepressant effects in the adult rodent brain. We fo- cused our studies to medial prefrontal cortex, a brain region associated in the path- ophysiology of depression and antidepressant actions. Specifically, mice received continuous 50% of N>O for an hour after which the animals breathed room air for another hour. This treatment readily increased the expression of mRNAs (c-fos, arc, badnf, zif-268, homer-1A, egr-2, mkp-1, synapsin) connected with cortical excitability and rapid-acting antidepressant effects (Fig. 1A). Unexpectedly, however, phos- phorylation of TrkB and phosphorylation of GSK3 into the inhibitory serine-9 resi- due remained unaltered (Fig. 1A). When samples were collected from mice eu- thanized during N2O administration similar observations were seen, indicating that ongoing NMDA-R blockade is not inherently coupled with TrkB and GSK3 signaling alterations (Fig. 1B). Phosphorylation of mitogen-activated protein kinase (MAPKT202/¥204) and expression of activity-dependent immediate early genes (IEGs), however, were increased during N2O administration (Fig. 1B-C), which confirms the immediate “excitatory” effects under NO. Notably, these changes induced by NO resemble those produced by the electroconvulsive therapy (Dyrvig et al. 2014,

Gene. 539, 8—14; Liet al. 2010, Science. 329, 959-964), and sleep deprivation (Ci- relli et al. 1995, J. Sleep Res. 4, 92—106), which also rapidly alleviates depression in a subset of patients.

Unlike ketamine HNK acts only as a weak NMDA-R antagonist (Suzuki et al. 2017,

Nature. 546, E1-E3) and is thus devoid of psychotomimetic and anesthetic proper-

S ties even at high doses (Zanos et al. 2016, Nature. 533, 481—486). Instead, HNK

N facilitates AMPA-R function, which is considered as its main pharmacological action & (Zanos et al. 2016, Nature. 533, 481-486). To investigate whether AMPA-R activa- n 30 tion regulates TrkB and GSK3B phosphorylation, we subjected mice to HNK and

I ketamine treatments. The phosphorylation levels of TrkB and GSK3B remained, = however, unaltered 30 min after HNK injections (Fig. 2A). More intriguingly, suban- i esthetic ketamine produced also only minor acute phosphorylation changes on TrkB

R and GSK3B (Fig. 2A-D). The phosphorylation of p70S6k™21/8424 a kinase down- > 35 stream of the TrkB-mTor pathway, also remained unchanged by these treatments (Fig. 2A-D). In contrast, and more unexpectedly, the ability of ketamine to acutely regulate these molecular events increased dose-dependently and most significant effects were observed with anesthetic doses (Fig. 2A-D-3A). Notably, an anesthetic dose of ketamine increased phosphorylation of TrkB, p70S6k and GSK3 within 3 min when its metabolism into HNK is likely marginal (Fig. 3B). Most importantly, a sedative dose of ketamine deuterated at the C6 position, a modification that reduces its metabolism into HNK (Zanos et al. 2016, Nature. 533, 481-486), recapitulated the acute effects of equivalent dose of ketamine on TrkB and GSK3B phosphoryla- tion (Fig. 2B). Collectively these data indicate that the ability of ketamine to acutely regulate TrkB and GSK3B signaling is by no means restricted to subanesthetic doses and that HNK is not responsible for these effects of ketamine.

To provide further insights for the intriguing differential responses of subanesthetic versus sedative-anesthetic doses of ketamine and apparent lack of the acute effects of HNK and N20 on TrkB and GSK3B signaling we performed time-lapsed guantita- tive pharmaco-EEG recordings in freely moving mice subjected to the treatments.

Ketamine increased high frequency gamma oscillations in a dose-dependent man- ner (Fig. 2C-D, 4). Low and high ketamine doses produced, however, different acute changes within the lower EEG frequencies. While doses <10 mg/kg produced no clear alterations, higher doses increased frequencies between ~1-5 Hz (delta, low theta) and between ~20-50 Hz (beta, low gamma) and reduced ~10 Hz (alpha) (Fig. 2C-D,4). Other than a slight increase in alpha and gamma oscillations HNK did not produce clear acute alterations in EEG spectra (Fig. 2D). Apart from slight damp- ening of low gamma oscillations (and initial dampening of delta oscillations), no ma- jor sustained EEG alterations was observed during N2O administration (Fig. 1, 5).

Further, we analyzed the EEG post-N>O to see whether such evoked slow EEG oscillations in response to preceding N2O exposure can be recapitulated in rodents.

Indeed, EEG oscillations at the range of ~1-5 Hz gradually, yet transiently, emerged

S above baseline upon withdrawal from an hour exposure to 50% N20 (Fig. 1D). No-

N tably, increased slow EEG oscillations appeared rapidly after a short exposure to & higher concentrations of NO (Fig. 1E, 5A-B). Beta and low gamma oscillations n 30 were reduced during this N2O treatment but these alterations rapidly normalized

I upon gas withdrawal (Fig. 1D-E, 5A-B). Altogether these data prompted us to collect = brain samples for western blot analyses during these recovery periods after expos- i ing the animals to varying concentrations of NoO. Remarkably, phosphorylation of

R TrkB, GSK3B and p70S6k were up-regulated in these samples, while most promi- > 35 nent changes were seen with 65% N>O (Fig. 1F, Fig. 6). Collectively these data demonstrate that N>O can indeed regulate TrkB and GSK3B signaling in the brain but these responses appear only after gas withdrawal during which slow EEG oscil- lations become facilitated.

Observations obtained with NoO guided us to test whether ketamine might evoke similar EEG responses at subanesthetic doses that are shown to increase cortical excitability and to bring rapid antidepressant effects (Autry et al. 2011, Nature. 475, 91-95; Li et al. 2010, Science. 329, 959-964). While a low dose of ketamine again failed to produce acute increase in slow EEG oscillations, these oscillations emerged gradually and only after the peak of pharmacological effect of ketamine (Fig. 1G, 7). Interestingly, gamma and slow-wave delta oscillations showed inverse time-dependent regulation after ketamine (Fig. 1G, 7). These effects of subanes- thetic N2O and ketamine on EEG resemble, but are qualitatively quite different, to those produced by flurothyl-induced seizure (Fig. 1H), another historical treatment of depression (Krantz et al. 1957, Science. 126, 353-354). Slow-wave delta and theta oscillations emerged rapidly after flurothyl withdrawal while other overshooting

EEG oscillations were not noted. Phosphorylation levels of TrkB, GSK3B and p70S6k were, however, significantly increased during the post-ictal period (Fig. 1H), further demonstrating that slow EEG oscillations predict these signaling responses.

Direct facilitation of slow EEG oscillations and TrkB signaling without preced- ing cortical excitability is not translated into antidepressant responses

Slow EEG oscillations are characteristic for sedation and reduced vigilance states and are induced by drugs carrying sedative properties. Indeed, we have previously shown that conventional antidepressants, particularly tricyclics that block H4-R (his- tamine-1 receptors), activate TrkB within similar time frame as ketamine (Rantamäki

S et al. 2007, Neuropsychopharmacol. 32, 2152—2162; Saarelainen et al. 2003, J.

N Neurosci. 23, 349-357), albeit this controversy has received little attention (Rantamäki and Yalcin, 2016, Prog. Neuropsychopharmacol. Biol. Psychiatry. 64, n 30 285-292). To test the intriguing possibility that mere sedation co-associates with

I increased TrkB and GSK3B phosphorylation changes we injected mice with a hyp- = notic-sedative drug medetomidine (an a2-noradrenergic receptor agonist) that spe- i cifically increase slow EEG oscillations (Fig. 8A-B). Notably, while medetomidine

R readily regulates TrkB and GSK3B signaling it concomitantly dampens > 35 MAPKT202/¥204 phosphorylation and gamma oscillations (Fig. 2B, 12). Moreover, if anything medetomidine reduces IEG expression (Fig. 11).

Our report links ongoing slow EEG activity with some of the key molecular signaling events connected with rapid antidepressant effects. The differential mechanistic principles underlying the abilities of rapid-acting antidepressants, conventional anti- depressants and hypnotic-sedative drugs to regulate slow EEG oscillations and thereby TrkB and GSK3B signaling suggests that activation and inhibition of TrkB and GSK3B, respectively, are not per se sufficient for rapid antidepressant re- sponses. We tested this hypothesis with medetomidine in the learned helplessness paradigm, which has strong construct validity regarding depression (Vollmayr and

Henn, 2001, Brain Res. Brain Res. Protoc. 8, 1-7). In this model a rodent is exposed to inescapable mild foot shocks and subsequently tested for a deficit (helplessness) of acquired avoidance. Subanesthetic dose of ketamine rapidly (within 24 h) ame- liorated the avoidance deficit while medetomidine showed no effect (Fig. 8D). Col- lectively our data support a notion that consecutive regulation of cortical excitability and regulation of TrkB and GSK3B during the rebound slow EEG oscillations is shared neurobiological phenomenon for interventions that can bring rapid antide- pressant responses in humans. This hypothesis is supported by clinical studies with

ECT. Indeed, rather than mere seizure manifestation, post-ictal (i.e. after seizure) emergence of slow EEG oscillations have been associated with the efficacy and onset of action of ECT (Nobler M. S. et al., 1993, Biol. Psychiatry 34, 321-330;

Sackeim H.A. etal, 1993, N. Engl. J. Med. 328, 839-846) (see Fig. 1H).

Therapeutic efficacy of rapid-acting antidepressants may be determined by utilizing slow neural oscillations

Results of the present study are summarized in Figures 13 and 14. The present disclosure proves that by monitoring slow neural oscillations from the cortex of the

S brain of a subject administered with one or more rapid-acting antidepressant(s) by

N electrophysiological monitoring, it is possible to determine the therapeutic efficacy & of said rapid-acting antidepressant(s) based on fluctuations on slow neural oscilla- n 30 tions before the administration and during E phase (excitation) and | phase (inhibi-

I tion) after the administration under the influence of said rapid-acting antidepres- = sant(s) in said subject. In a specific embodiment the electrophysiological monitoring i revealing more slow oscillations in the "I phase” compared to the “E phase” indicates

R the therapeutic efficacy. In a very specific embodiment the electrophysiological mon- > 35 itoring revealing at least 5%, 10%, 15% or more slow oscillations in the "I phase” compared to the “E phase” indicates the therapeutic efficacy or outcome of the rapid-acting antidepressant.

In a specific embodiment any treatment, which produces sufficient rebound inhibi- tion in the cortex possess rapid antidepressant effects. Inhibition can be monitored using e.g. EEG/MEG (slow neural oscillations: 1-6 Hz) and/or any other physiologi- cal mean correlated with the emergence of aforesaid changes. In another specific embodiment any intervention transiently (e.g. 1 s — 2 h) facilitating brain excitability, which produces sufficient rebound inhibition in the cortex, possess rapid antidepres- sant effects. Sufficient inhibition can be monitored using e.g. EEG/MEG (slow neural oscillations: 1-6 Hz (e.g. 5 %, 10 %, 15 % or more slow oscillations in the | phase compared to the E phase)) and/or any physiological mean or clinical evaluation cor- related with the emergence of aforesaid changes.

Figure 13 shows the essential interplay between “excitation” (E phase) and “inhibi- tion” (| phase) caused by rapid-acting antidepressants. Rapid-acting antidepres- — sants produce cortical excitability that evokes a homeostatic emergence of slow neural oscillations, during which molecular events intimately implicated with rapid antidepressant effects become altered: activation of TrkB receptor and inhibition of

GSK3B (glycogen synthase kinase 3B). Such evoked homeostatic brain responses beneficial against depression can be rapidly produced and reproduced and con- trolled with interventions capable of producing transient cortical excitability. Monitor- ing of the time-lapsed emergence of slow wave neuronal network oscillations during the treatment(s) can be utilized to control antidepressant efficacy. All figures 1-12 and 14, especially e.g. figures 1 and 8, support figure 13.

Figure 14 illustrates three example schemes (in a time line) enabled by the present invention. Scheme 1 describes a method for determining a therapeutic efficacy of a

S rapid acting antidepressant by monitoring slow neural oscillations. Desired altera-

N tions of slow neural oscillations reveal the presence of therapeutic effects (e.g. re- & bound oscillations or more slow neural oscillations in the I phase compared to the E n 30 phase). Allfigures 1-14, especially e.g. figures 1 and 8, and figures 2-3, 5-6, support

I Scheme 1. Schemes 2 and 3 describe a real time method for optimizing rapid acting = antidepressant treatment by monitoring slow neural oscillations. If a desired re- i sponse is not achieved with a rapid acting antidepressant treatment said treatment

R may be e.g. repeated (e.g. figure 1, 5) or modified (e.g. adjusting dose) (e.g. figure > 35 1) orreplaced with another rapid acting antidepressant in order to arrive at a desired or improved outcome (e.g. more slow neural oscillations in the | phase compared to the E phase). All figures 1-14, especially e.g. figure 4, support Schemes 2 and 3.

Schemes 1-3 are also applicable e.g. for methods of screening novel rapid acting antidepressants or combinations thereof.

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Claims (16)

Claims

1. A method for determining a therapeutic efficacy of a rapid-acting antidepressant, characterized in that the method comprises: real-time monitoring of slow neural oscillations from the cortex of the brain of a subject by electrophysiological monitoring administered with one or more rapid- acting antidepressant(s), and determining a therapeutic efficacy of said rapid-acting antidepressant(s) based on comparing fluctuations on slow neural oscillations at baseline (before the admin- istration) and during the influence of said rapid-acting antidepressant(s) in E phase (excitation) and thereafter in | phase (inhibition) in said subject.

2. The method for determining a therapeutic efficacy of a rapid-acting antidepres- santofclaim1, characterized in that the therapeutic efficacy of the rapid-acting antidepressant(s) is determined by more slow oscillations in the | phase compared to the E phase.

3. The method for determining a therapeutic efficacy of one or more rapid-acting antidepressants of any one of claims 1 - 2, characterized in that duration of the E phase is 1 second — 2 hours and/or duration of the | phase is 5 min — 1 hour and/or duration of the combination of E and | phases is 5 min — 3 hours.

4. The method for determining a therapeutic efficacy of one or more rapid-acting antidepressant(s) of any one of claims 1 — 3, characterized in that the electro- o physiological monitoring is an electroencephalogram (EEG) and/or magne- S toencephalography (MEG) and/or other mean. 3 n

5. The method for determining a therapeutic efficacy of one or more rapid-acting Ir 30 antidepressant(s) of any one of claims 1 — 4, characterized in that slow neural E oscillation frequency bands comprise or have the frequency range 1 — 6 Hz. S

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6. The method for determining a therapeutic efficacy of one or more rapid-acting > antidepressant(s) of any one of claims 1 — 5, characterized in that the thera- peutic efficacy of the rapid-acting antidepressant(s) is determined by concurrent emergence of E phase and | phase.

7. The method for determining a therapeutic efficacy of one or more rapid-acting antidepressant(s) of any one of claims 1 — 6, characterized in that the thera- peutic efficacy is for nervous system disorder associated with compromised plastic- ity, e.g. a disorder is selected from the group consisting of depression, anxiety, ad- diction, neurodegenerative disorder, brain trauma, post-traumatic stress disorder and neuropathic pain.

8. The method for determining a therapeutic efficacy of one or more rapid-acting antidepressant(s) of any one of claims 1 — 7, characterized in that the rapid-acting antidepressant is a pharmacological compound that has one or more of the following properties: NMDA-R blockade (e.g. ketamine, nitrous oxide), GABAA-R blockade (e.g. flurothyl), GABA4-R positive allosteric modulation (e.g. gamma-hydroxybutyrate), GHB-R agonism (e.g. gamma-hydroxybutyrate), AMPA- R positive allosteric modulation (e.g. hydroxynorketamine), 5-HT2A-R agonism (e.g. psilocybin), alfa2-R antagonism (e.g. atipamezol), anti-muscarinic, up-regulate im- mediate-early genes, produce seizures, evoke glutamate release; or any related pharmaceutical antidepressant or any combination thereof, and/or the rapid-acting antidepressant(s) is(are) a non-pharmacological antidepressant se- lected from the group consisting of sleep deprivation, electroconvulsive therapy (ECT), (repetitive) transcranial magnetic stimulation (TMS), transcranial direct cur- rent stimulation (tDCS), vagal nerve stimulation, photic stimulation, direct current stimulation, hyperthermia, hypothermia, cortical cooling, or related physiological method, or any combination thereof and/or the rapid-acting antidepressants are a combination of one or more pharmacological rapid-acting antidepressants and one or more non-pharmacological rapid-acting antidepressants. S

N

9. The method for determining a therapeutic efficacy of one or more rapid-acting S antidepressant(s) of any one of claims 1 — 8, characterized in that the rapid- N acting antidepressant has been administered intravenously, intra-arterially, intra- E 30 — muscularly, intranasally, by an oral administration or by inhalation. N s

10. The method for determining a therapeutic efficacy of one or more rapid-acting = antidepressant(s) of any one of claims 1 — 9, characterized in that the rapid- i acting antidepressant has been administered on the same day when the therapeutic efficacy is determined.

11. The method for determining a therapeutic efficacy of one or more rapid-acting antidepressant(s) of any one of claims 1 — 10, characterized in that the method further comprises monitoring neurophysiological data, behavioral data, respiratory data, blood flow data, cardiac data, galvanic skin response data, data on biochemi- cal marker(s) or any combination thereof, specifically after the administration under the influence of said rapid-acting antidepressant(s) in said subject.

12. The method for determining a therapeutic efficacy of one or more rapid-acting antidepressant(s) of any one of claims 1-11, characterized in that the method does not comprise further monitoring.

13. The method for determining a therapeutic efficacy of one or more rapid-acting antidepressant of any one of claims 1 — 12, characterized in that said monitor- ing of the slow neural oscillations or determining the therapeutic efficacy is repeated once or twice or several times after the subject has further been administered with the rapid-acting antidepressant for the second time, third time or several times e.g. during the same day as the first administration, respectively, or after the subject has further been administered with another rapid-acting antidepressant.

14 The method for determining a therapeutic efficacy of one or more rapid-acting antidepressant of any one of claims 1 — 13, characterized in that said deter- mining is carried out in real-time.

15. The method for determining a therapeutic efficacy of one or more rapid-acting — antidepressant(s) of any one of claims 1 — 14, characterized in that TrkB and/or N GSKB signaling is(are) indirectly determined by monitoring slow neural oscillations A or sedative state of the individual. <Q N

16. A method of screening novel rapid-acting antidepressants, characterized in E 30 that the method comprises N real-time monitoring of slow neural oscillations from the cortex of the brain of = a subject by electrophysiological monitoring administered with a pharmaceutical, m and N determining a rapid-acting antidepressant therapeutic efficacy of said pharma- ceutical based on comparing fluctuations on slow neural oscillations at baseline (be- fore the administration) and during the influence of said pharmaceutical in E phase (excitation) and thereafter in | phase (inhibition) in said subject.

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