Effects of L-theanine on Motor Cortex Excitability in Healthy Subjects: A Paired-Pulse TMS Study

Effects of Single-dose L-theanine on Motor Cortex Excitability in Healthy Subjects: A Double-blinded, Randomized Order, Cross-over Paired-Pulse Transcranial Magnetic Stimulation Study

Major depressive disorder (MDD) is a serious mental illness and the leading cause of disability worldwide. New pharmacotherapeutic agents with complementary neurobiological mechanism and better side effect profile are of great needs. In addition to the monoamine system, the glutamatergic system plays a crucial role in MDD. L-theanine (N5-ethyl-L-glutamine) is the primary psychoactive component uniquely in green tea. Preclinical studies have demonstrated anti-depressant effect of L-theanine in rodents and provided evidences for its pharmacological properties of N-methyl-D-aspartate (NMDA) and gamma-aminobutyric acid (GABA) agonism. Yet these effects have not been proven in humans. Only one open-label clinical trial has studied and supported antidepressant effects of L-theanine in MDD patients. We propose using pair-pulse transcranial magnetic stimulation (ppTMS) to probe how L-theanine may manipulate the glutamatergic and GABA systems in the frontal region by changing cortical excitability first in healthy subjects. We plan to investigate the neurobiological effects of L-theanine in healthy subjects first. Granted that the first phase pilot trial provides neurophysiological evidence of L-theanine on motor cortex excitability in human subjects, next phases of studies on L-theanine in MDD patients cortical excitability could be justified.

Background and Significance: Major depressive disorder (MDD) is a serious mental illness and the leading cause of disability worldwide. Although many antidepressants acting on synaptic monoamine levels have been used as the first-line drug treatment for MDD, around one third of MDD are pharmacologically resistant. Side effects of these medications impose additional hardship on adherence and further affect treatment outcome. New pharmacotherapeutic agents with complementary neurobiological mechanism and better side effect profile are of great needs. In addition to the monoamine system, the glutamatergic system plays a crucial role in MDD. L-theanine (N5-ethyl-L-glutamine) is the primary psychoactive component uniquely in green tea. Epidemiological studies support that green tea consumption is an independent factor associated with lower prevalence of depression. Preclinical studies have demonstrated anti- depressant effect of L-theanine in rodents and provided evidences for its pharmacological properties of N-methyl-D-aspartate (NMDA) and gamma-aminobutyric acid (GABA) agonism. Yet these effects have not been proven in humans. Only one open-label clinical trial has studied and supported antidepressant effects of L-theanine in MDD patients. We propose using pair-pulse transcranial magnetic stimulation (ppTMS) to probe how L-theanine may manipulate the glutamatergic and GABA systems in the frontal region by changing cortical excitability first in healthy subjects. ppTMS is a well-established technique to investigate frontal motor cortical excitability mediated by the inter-neuron NMDA and GABA receptors. Specific changes of ppTMS measures, including impaired short-term and long-term intracortical inhibition (SICI, mediated by GABA-A receptor; LICI, mediated by GABA-B receptor) and intracortical facilitation (ICF, mediated by NMDA receptor), have been demonstrated in MDD. Using this technique, we plan to investigate the neurobiological effects of L-theanine in healthy subjects first. Granted that the first phase pilot trial provides neurophysiological evidence of L-theanine on motor cortex excitability in human subjects, next phases of studies on L-theanine in MDD patients cortical excitability could be justified. This will lay foundation for further exploration of L-theanine's potential as an augmenting agent for MDD in a placebo- controlled design. Aims and Hypothesis: Given the potential NMDA and GABA agonistic effects of L-theanine, we hypothesize that it increases intracortical inhibition and facilitation through enhancement of NMDA- and GABA-receptor mediated neurotransmission, in healthy subjects (N=10 to complete study). Study Procedures: Double-blinded, Randomized-order, Cross-over placebo-controlled to evaluate acute effect of single-dose L-theanine on motor cortex excitability by ppTMS in 10 healthy subjects. Dose of L-theanine or placebo is 400mg. At baseline, subjects will be randomized to L- theanine or placebo group, then receive ppTMS protocol before drug administration. The ppTMS protocol is repeated after 30min of administration. Then subjects will return to clinic after 1 week free of any medications and repeat the above protocol with the second drug condition. Visual analog scale will be used to evaluate psychosomatic symptoms and wellbeing of the subjects pre- and post-each drug administration. Data Analytic Plan: Wilcoxon test will be used to compare the baseline-to-post-drug means of SICI, LICI and ICF measures. Prespecified covariates include age, sex, handedness, level of fatigue will be analyzed in linear regression model. The time-condition relationship with continuous dependent variables of ICI and ICF values will be evaluated by Mixed Effect Model. Two- sided P value < 0.05 is considered statistically significant.

This is a pilot study, investigating the effects of single dose L-theanine in Healthy subjects, to further assess the validity and feasibility to study this compound in patient population.

Subject will receive 400mg single dose of L-theanine, by oral ingestion with water. The capsules are prepared and dispensed by hospital pharmacy, with the investigator and participant both blinded.

Subject will receive 400mg single dose of matching Placebo, by oral ingestion with water. The capsules are prepared and dispensed by hospital pharmacy, with the investigator and participant both blinded.

The subject will receive paired-pulse TMS (ppTMS) procedure before and 30min after taking the drug orally, to assess motor cortex excitability, measured by surface electromyogram (EMG). The ppTMS procedure is administered by a TMS stimulator controlled a program software named Signal. The coil of the stimulator is placed above the scalp where the stimulation would activate the left primary motor cortex region that controls the right thumb. When a pulse stimulation is delivered by the coil, the EMG over a thumb muscle (abductor pollicis brevis) will record a motor-evoked potential on the tracing. Cross-over with placebo in two separate sessions at least 72 hours apart.

The subject will receive paired-pulse TMS (ppTMS) procedure before and 30min after taking the drug orally, to assess motor cortex excitability, measured by surface electromyogram (EMG). The ppTMS procedure is administered by a TMS stimulator controlled a program software named Signal. The coil of the stimulator is placed above the scalp where the stimulation would activate the left primary motor cortex region that controls the right thumb. When a pulse stimulation is delivered by the coil, the EMG over a thumb muscle (abductor pollicis brevis) will record a motor-evoked potential on the tracing. Cross-over with L-theanine in two separate sessions at least 72 hours apart.

The changes of Short-interval Intracortical Inhibition (SICI), Intracortical Facilitation (ICF), and Long-interval Intracortical Inhibition (LICI) before and 30 minutes after each drug administration. SICI, ICF and LICI are paired-pulse TMS (ppTMS)-EMG outcome measures that assess the activity of motor cortex GABA-A, NMDA and GABA-B interneurons, respectively. They are measured by the ratio between the peak-to-peak amplitude of motor-evoked potential (MEP) elicited by a testing TMS pulse (120% of the intensity of the resting motor threshold, following a conditioning pulse at different inter-stimuli interval, 2-5 milliseconds for SICI, 10-20 milliseconds for ICF, 100-200 milliseconds for LICI) and the peak-to-peak MEP amplitude elicited by a single pulse (120% of the intensity of the resting motor threshold). The baseline-to-post-drug change of SICI, ICF and LICI elicited by L-theanine will be compared to that elicited by placebo within each subject.

Before and 30 minutes after each drug administration (no long-term follow up as this is a study on acute effect of a single-dose agent).

VAS is a quick scale to assess and track how the participant subjectively feels through out each study session, e.g. anxiety, depression, excitement, etc. The score for each word ranges from "0" being the least in your life and "100" being the most in your life. It takes 3-4 minutes to complete each VAS, and there are 4 takes of VAS during each session. The outcome measure is the change of VAS throughout the 4 time points: 1) before the baseline ppTMS procedure; 2) before drug administration; 3) 30 minutes after drug administration; 4) before discharge from the session, during the session.

Throughout each session; each session lasts up to 3 hours; 2 sessions for each subject. The 2 sessions are 3-7 days apart.

Inclusion Criteria: Adult, aged between 18 and 65 years old; Able to read/speak English and give informed consent No current or history of Axis I psychiatric disorders by DSM-5. Free of psychotropic medication use Exclusion Criteria: History of significant acute or chronic neurological or medical disorder or condition that increases risk for seizure with TMS; History of alcohol use disorder, nicotine dependence, adjustment disorder; History of allergic reactions to L-theanine or green tea; Pregnancy; Unable/unwilling to abstain from nutraceutical supplements and psychotropic agents during participation in the study Unable/ unwillingness to refrain from recreational substance use (e.g. alcohol or marijuana) during participation in the study; Meet criteria for exclusion from TMS or MRI procedures, including intracranial metal implants or nonremovable ferromagnetic items in the head/neck.

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