William Shell, MD, Debbie Bullias, B.S, Elizabeth Charuvastra, RN, Larry May, MD and David Silver, MD

Introduction

The management of sleep disorders continues to be a treatment challenge because hypnotic drugs have therapeutic limitations and dose related side effects. Hypnotic drugs may abolish stage IV sleep, reduce REM sleep, contribute to morning grogginess, or induce residual cognitive dysfunction. Tolerance and long-term dependency also occur. Medications such as tricyclic antidepressants and antiepileptics produce less dependency but have other side effects such as dry mouth and dizziness. The inherent difficulties with these drugs are magnified by the large number of people who suffer from sleep disorders. Even a small incidence of side effects is significant when the population at risk is large.

Certain amino acids are precursors to the neurotransmitters known to influence sleep quality including sleep latency, duration of sleep, and REM sleep. The use of amino acids to produce neurotransmitters that affect sleep cycles has been handicapped by the large dose requirement for individual amino acids, the attenuation of response, and the potential side effects of high dose amino acids. An amino acid formulation that can stimulate neurotransmitters that regulate normal sleep cycles without the known problems could provide an alternative to the hypnotic drugs currently in use.

GABAdone is an amino acid based formula that contains 5-hydroxytryptophan, GABA, and low dose choline bitartrate. The 5-hydroxytryptophan is a tryptophan analog believed to influence both sleep latency and sleep maintenance by modifying serotonin levels. GABA is known to reduce sleep latency. Choline supports acetylcholine synthesis which potentiates bursts of REM sleep while facilitating stage IV sleep. Previous studies have examined the use single amino acids to influence sleep quality but combinations have not been evaluated.

This study was a randomized, double blind, placebo controlled trial of GABAdone in patients with certain sleep disorders. The end points of the study included ability to fall asleep and assessment of sleep quality. In addition, a 24-hour ECG measurement of heart rate variability was used to objectively assess the influence of the autonomic nervous system on sleep cycles.

Study Design:

A total of 18 subjects were randomized to treatment and placebo. Each of the 18 subjects underwent baseline examination to include sleep quality questionnaires and 24- hour electrocardiographic recording. Nine subjects were randomized to a one week ingestion of GABAdone at bedtime and nine subjects were randomized to a one week ingestion of placebo at bedtime. Each morning after ingestion of either active or placebo, the subject filled out sleep questionnaires including both visual analogue scales and Likert Scale formats. On the sixth day, a repeat 24-Hour ECG examination to include the seventh night was performed in all 18 subjects. Baseline and day 7 ECG data were analyzed.

Patient Inclusion and Exclusion:

Subjects were identified by solicitation of persons interested in taking a non-addicting formula that would improve the quality of sleep. Patients were included if they were over the age of 18, below age 65 and had a history of intermittent non-restorative sleep.

The exclusions included subjects who were currently taking sleeping medication, previous GABAdone use, known endocrine disease, pregnant or lactating females and subjects with implanted electrical devices.

Sleep Protocol:

The subjects were asked to maintain their current sleep program.

Data Collection:

At baseline the following measurements were taken: weight, percent body fat, age, sex, Pittsburgh Sleep Quality Index (PSQI) and Leeds Sleep Evaluation Visual Analogue Scale (LSEQ). On each of day one through seven the PSQI and LSEQ were obtained in the morning after awakening. On day seven the LSEQ and PSQI were filled out and the patient returned to the clinic. The forms were obtained and baseline measurements were repeated. On day six, a second 24-hour ECG recording was begun and completed on day seven. The amino acid formulation or placebo was taken on day one through seven.

Visual Analogue Scale Measurement of Sleep Quality the Leeds Sleep Evaluation Questionnaire (LSEQ):

The LSEQ Sleep Valuation Questionnaire (LSEQ) has been specifically designed to monitor subjectively perceived changes in sleep duration. The LSEQ consists of a 100mm visual analog scale (VAS). Four factors were measured on each LSEQ including: 1. Time to fall asleep 2. Quality of sleep 3. Ease of waking from sleep, and 4. Behavior following awakening. Each LSEQ was compared to the subjects’ usual sleep pattern. A lower score indicated better quality of sleep.

Pittsburgh Sleep Quality Index (PSQI):

The PSQI is a validated self-reported questionnaire that provides specific sleep related information and a rating of sleep related factor on a Likert Scale. The specific sleep information included bedtime, rising time, minutes to fall asleep and actual hours slept. We also collected the number of awakenings per night. The subjective sleep quality indices included quality of sleep, daytime alertness, nighttime awakenings, respiratory distress, physical discomfort, frequency of dreams, disturbing dreams, perception of snoring, and daytime dysfunction. For each subject a total Likert score was generated.

The data analyzed from the PSQI included duration of sleep, total amount of sleep, sleep efficiency and subjective sleep quality. On day seven, daytime dysfunction was analyzed.

The subjects were blinded to the nature of the capsule at the time the LSEQ and PSQI questionnaires were administered. The data were entered before the codes were broken

24Hour ECG Recording:

Patients underwent continuous recording of the 24-hour electrocardiogram using a Delmar/Reynolds Lifecrad CF ECG recorder. The ECG data was collected in digital format using a three lead hook-up that generated three channels of ECG data. The ECG data was analyzed on a Delmar/Reynolds’ Playback System (Pathfinder 700 Series) for arrhythmia and heart rate variability. The heart rate variability analysis was performed in both the time and frequency domains according to the standards published by the Task Force of The European Society of Cardiology and The North American Society of Pacing and Electrophysiology. Both global 24-hour and sleeping hours circadian activation of parasympathetic function were measured.

Amino Acid Formulation: GABAdone contains choline bitartrate, 5-hydroxytryptophan as griffonia extract, GABA, grape seed extract, hydrolyzed whey protein, valerian extract, ginkgo biloba, glutamic acid, and cocoa. A two capsules dose contains a total of 2000 mg.

The primary end points were the difference in sleep latency, duration of sleep, and perceived AM grogginess and changes in autonomic nervous system function.

Statistical Analysis:

The PSQI questionnaires were measured using a centimeter ruler to obtain continuous data. From this continuous data mean, standard deviation and standard error could be computed. The data was analyzed with Student t-tests. Mean differences were also analyzed with Student t-tests without correction for normality.

The PSQI data were not continuous but included a five-point scale. The data were averaged and mean and standard deviation computed. Student t-tests were applied to the data. Mean differences were computed and analyzed without normality correction. This is a frequently used method for analysis of non-continuous Likert scale data. Normality corrections are not used to analyze Likert Scale data.

The data obtained from the 24-hour ECG analysis was continuous data and were analyzed with Student t-test.

Results:

Time to Fall Asleep—Sleep Latency

In the active group, the baseline time to fall asleep was 32.3 minutes compared to 19.1 minutes after GABAdone administration (t=2.91, p<0.01, n=9). In the placebo group, the baseline time to fall asleep was 34.8 minutes compared to 33.1 minutes after placebo administration (p, ns, n=9). The difference was statistically significant (p<0.01) (Figure 1).

Hours Slept

In the active group, the duration of sleep was 5 hours at baseline, compared to 6.83 hours after GABAdone administration (t=-3.14, p<0.01, n=9). In the placebo group, the baseline duration of sleep was 7.16 hours compared to 7.11 hours after placebo administration (p, ns, n=9). The difference was statistically significant (p<0.01).

Ease of Falling Asleep

The ease of falling asleep was measured using a visual analog scale. In the active group, the baseline perceived ease of falling asleep 44.4 mm on a 100 mm scale compared to 64.8mm after GABAdone administration (p=0.02, n=9). In the placebo group, the perceived ease of falling asleep was 43mm at baseline, compared to 54mm following placebo administration (p=0.11, n=9). The differences were not statistically significant. (Figure 2)

Awakenings

In the active group, the baseline number of perceived awakenings was 4.3 compared to 2.6 after GABAdone administration (p<0.01, n=9). In the placebo group, the baseline awakenings were 2.8 compared to 3.1 (p, ns) after placebo administration. The difference was statistically significant (p=0.02, n=18). (Figure 3)

Minutes Awake During Awakenings

In the active group, the number of minutes during the awakenings was perceived as 21.1 minutes during baseline compared to 8.3 minutes following GABAdone administration (p=0.02, n=9). In the placebo group, the number of minutes during awakenings was perceived as 46.9 minutes at baseline compared to 36.7 (p, ns) following placebo administration (Figure 4)

Restorative Sleep and AM Grogginess

As a measure of restorative sleep, we assessed perceived AM grogginess using a 100mm Visual Analog Scale (VAS). In the active group, the perceived AM grogginess was 30.6mm at baseline was 30.6mm compared to 11.1mm after GABAdone administration (p<0.01, n=9). In the placebo group, the perceived AM grogginess was 67.8 mm at baseline, compared to 65 mm after placebo administration (p, ns n=9).

Autonomic Nervous System Function

Assessment of parasympathetic autonomic nervous system function, an objective measure of nighttime autonomic function, showed substantial improvement compared to placebo. There were no adverse events reported in either the treatment or placebo groups.

Discussion

The carefully timed activation of the three neurotransmitters–serotonin, acetylcholine, and GABA—is required to initiate sleep, maintain sleep, induce restorative sleep, and increase the amount and duration of REM sleep. If the timing and secretion of these three neurotransmitters are altered, normal sleep cycles and restorative sleep do not occur. For example the benzodiazepine drugs reduce sleep latency but abolish phase IV- V sleep and REM sleep.

The release of serotonin initiates sleep and reduces measured latency. The timing of serotonin release is critical to initiation of sleep. The amount of serotonin released is also critical to initiation of sleep. At the initiation of sleep, a small amount of serotonin is released. The peak concentration of serotonin occurs within several hours after sleep initiation. The failure to produce serotonin, the production of insufficient serotonin, or the production of excessive amounts of serotonin will result in the failure to initiate sleep.

Production of acetylcholine after initiation of sleep results in restorative Delta IV-V sleep. Following the burst of serotonin that initiates sleep, acetylcholine release increases the duration of phase IV and V restorative sleep. Acetylcholine production in the sleep centers increases the frequency and duration of REM episodes. The commonly used hypnotics abolish phase IV and V sleep and inhibit REM sleep.

GABA is the main inhibitory neurotransmitter. The initiation and maintenance of sleep also depends on the availability of GABA to the GABA-receptors. The most commonly used drug hypnotics act by sensitizing the GABA receptors to GABA. GABA provides general inhibition of the nervous system that allows sleep to be initiated and maintained.

GABAdone contains a formula blend of selected GRAS (generally regarded as safe) ingredients that are derived from the normal human food chain. The primary ingredients are key amino acids. The GABAdone formula is designed to increase the function of the neurotransmitters serotonin, acetylcholine, and GABA in patients with sleep disorders. The GABAdone formula is based on a five-component, patent pending process to provide for the conversion of a neurotransmitter precursor into a neurotransmitter. The five-component system includes: (1) each neurotransmitter is synthesized from an amino acid precursor, (2) stimulation of the uptake of the neurotransmitter precursor is required to initiate the conversion of a precursor to a neurotransmitter, (3) since most neurons are inhibited from firing, an adenosine antagonist such as and cocoa powder is added to disinhibit the neuron, (4) stimulation of neurons to release a specific neurotransmitter is required, and (5) a system must be used to prevent attenuation of the precursor response, a well known precursor phenomena. GABAdone has been formulated to encompass this five-component system.

GABAdone is designed to produce neurotransmitters related to physiologic functions including initiation of sleep, maintenance of sleep, and re-induction of sleep if awakening occurs during the night. In the GABAdone formulation, choline is used as a precursor to acetylcholine. 5-hydroxytryptophan is utilized as a precursor to serotonin. GABA is directly administered as an inhibitory neurotransmitter. Ginkgo biloba is used as an uptake stimulator. Glutamic acid is used to produce glutamate, a neuronal stimulator. Cocoa is used to disinhibit the adenosine break. Grape seed extract, containing polyphenols, is used to avoid the attenuation usually associated with neurotransmitter precursor administration. GABA is administered as an inhibitory neurotransmitter.

Sleep disorders are associated with inadequate availability of the key neurotransmitter precursors choline, 5-hydroxytryptophan, and GABA. Determination of the Recommended Dietary Allowance (RDA) of a dietary ingredient in normal subjects is frequently accomplished by analyzing blood levels of the nutrient in normal subjects. Assessing the nutritional deficiency in the presence of a disease such as a sleep disorder is more complex. FDA scientists have proposed a physiologic methodology to determine the nutritional deficiency during a disease. If a physiologic parameter such as sleep latency is measured, administer the nutrient such as 5-hydroytryptophan and the physiologic parameter improves such as reduction of sleep latency, you have established the presence of the nutrient deficiency in the disease. These studies indicate that sleep disorders are associated with the nutrient deficiencies of choline, 5-hydroxytryptophan, and GABA.

This study supports the concept that combining neurotransmitter precursors in a precise, small dose ratio, may help in the management of sleep disorders. Larger, randomized trials to confirm the results seen here need to be performed.