DARK Classics in Chemical Neuroscience: Gamma-Hydroxybutyrate

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DARK Classics in Chemical Neuroscience: Gamma-hydroxybutyrate (GHB) Trevor A Trombley, Rory A Capstick, and Craig W Lindsley ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.9b00336 • Publication Date (Web): 09 Jul 2019 Downloaded from pubs.acs.org on July 17, 2019

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ACS Chemical Neuroscience

DARK Classics in Chemical Neuroscience: Gamma-hydroxybutyrate (GHB) Trevor A. Trombley1,¥, Rory A. Capstick1,¥, and Craig W. Lindsley1,2,3,4*

1Vanderbilt

Center for Neuroscience Drug Discovery, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, United States 2Department

of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232,

United States 3Department

of Chemistry, Vanderbilt Institute of Chemical Biology, Vanderbilt University, Nashville, Tennessee 37232, United States 4Department

of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232,

United States ¥These

authors contributed equally

TOC

Abstract Gamma-hydroxybutyrate (GHB) is a naturally occurring short-chain fatty acid that rose to prominence as a popular club drug in the 1990s. Originally developed as an anesthetic in the early 1960s, it was later sold as an over-the-counter dietary supplement before becoming a rising substance of abuse in the following decades as one of the ‘date rape’ 1 ACS Paragon Plus Environment

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drugs. Despite its abuse potential, there has been a recent surge in therapeutic interest in the drug due to its clinical viability in the treatment of narcolepsy and alcohol abuse/withdrawal. Its interactions with the GABAergic framework of higher mammals has made it the prototypical example for the study of the chief inhibitory mechanism in the human CNS. Though relatively obscure in terms of popular culture, it has a storied history with widespread usage in therapeutic, recreational (‘Chemsex’), and some disturbingly nefarious contexts. This article aims to capture its legacy through review of the history, synthesis, pharmacology, drug metabolism, and societal impact of this DARK classic in chemical neuroscience. Keywords: Gamma-hydroxybutyrate, GHB, substance abuse, GABA, narcolepsy Introduction O

O

O OH

HO

NH2

HO

1, GHB

+

OH

Na-O

2, GABA

3, sodium oxybate Me N

O O

OH

HO

O N

O 2N

F

4, GBL

5, BD

6, flunitrazeepam

MeN OH

O

O

O

HN Cl

O 7, scopolamine

8, ketamine

Figure 1. Structures of GHB (1), GABA (2), sodium oxybate (3), GBL (4), BD (5) and other ‘date rape’/club drugs 6-8.

Gamma-hydroxybutyrate (GHB, 1) is a short-chain fatty acid used for a variety of recreational and therapeutic purposes and is endogenously present in the human CNS. It can also be found in various exoneural organs and tissues, as well as some meats, 2 ACS Paragon Plus Environment

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fruits and wines, though often at concentrations too low to produce any noticeable pharmacological effects (Figure 1).1 It is most commonly encountered as a viscous colorless solution due to the strongly hygroscopic nature of its sodium salt, which is the most synthetically-accessible form of the drug.2 First used as an anesthetic in the early 1960’s, GHB has remained mostly out of the public eye, though its prevalence in club scenes, particularly those in Europe, has garnered significant attention over time.3 GHB is used for its depressant and euphoria-inducing effects, which are mediated by its binding to both Gamma-aminobutyric acid subtype B (GABAB) and GHB receptors within the CNS.4,5 Gamma Aminobutyric Acid (GABA, 2) serves as the primary depressive neurotransmitter in the CNS of higher mammals.6 Its receptor class consists of both ligand-gated ion channels (GABAA) and G-protein coupled receptors (GABAB).7,8 GHB’s interaction with the GABAB receptor has been well-characterized, and is responsible for the depressive effects of the drug.1,4,9 Its binding causes G-protein-dependent inwardlyrectifying potassium (GIRK) channels to open, resulting in hyperpolarization of the cell, and a reduction in neurotransmitter release.10 Behaviorally, this results in depressive, alcohol-like symptoms such as loss of inhibition, improved sociability, ataxia, and drowsiness.5,11 It is classified as a schedule 1 drug in the US, but some formulations including XyremTM (sodium oxybate, 3) have been approved by the Food and Drug Administration (FDA) as clinical treatments for narcolepsy.2 Other formulations of sodium oxybate have also been used to treat severe alcoholism (AlcoverTM), though this practice is less common today.12 During its peak popularity, GHB was frequently used in European clubs due to its widespread availability as an over-the-counter weight loss supplement. Its characterization as a club drug may be slightly suspect given its widespread use in domestic settings, though this is probably due to the drug’s reputation in popular culture rather than recorded data.13 Its ubiquity in club scenes, combined with growing concern over its negative side effects including seizures and respiratory failure led several European countries to restrict the sale of GHB in the early 1990s. Despite these regulations, illicit forms of the drug were easily synthesized well into the 2000s due to the accessibility of its precursors Gamma-butyrolactone (GBL, 4), thanks to its use in industrial paint thinners, as well as in the production of the chemical solvent N-Methyl-2pyrrolidinone (NMP), and 1,4-butanediol (BD, 5).14,15 GHB, ‘liquid ecstasy’, is notorious as one of several ‘date rape’ drugs, also referred to as ‘club drugs’ including flunitrazeepam (rohypnol, roofies) (6), scopolamine (7), ketamine, ‘special K’, (8) in the form of liquids, pills and powders.16 Prevalent in the popular press, the dose-dependent CNS effects of GHB from euphoria to respiratory depression to unconsciousness, and in higher doses, coma and death earn it the status of a DARK Classic.

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GHB is trivially accessible from GBL via hydrolysis only requiring a clandestine chemist to have access to sodium hydroxide (lye) and heat. This process was only made easier by the online sale of GBL kits including complete instructions and ingredients, allowing anyone who ordered the ability to synthesize GHB on a several-hundred-gram scale with ease.17 As a result of this simplicity of synthesis, GBL was made a schedule 1 controlled substance in the US with the passing of the Date-RAPE Drug Prohibition Act on April 24th, 2000.18 Many other countries followed suit by restricting the sale of GBL in the following years.19-21 Table 1. Legal status of GHB and GBL in various western countries.19-21

Country United States Canada European Union

GHB Legality Restricted (Schedule I) Restricted (Schedule I) Restricted (Schedule IV)

United Kingdom Norway

Restricted (Schedule C) Restricted (License Required) Restricted (Schedule I)

Sweden

GBL Legality Restricted (Section 1) Restricted (Section 1) Unrestricted (Voluntary monitoring list) Restricted (Class C) Restricted (License Required) Restricted (Schedule I)

Table 1 summarizes the current legal statuses of GHB and its common precursor GBL in several western countries. Although GHB is restricted in all countries listed, access to GBL varies from country to country. It is considered to be under voluntary monitoring by the European Union, with countries like Norway allowing its sale given the proper licensing. Poland has no restrictions on the sale of GBL, as the prodrug is commonly sold as a chemical solvent.19 Though this offers home chemists one way to synthesize GHB via the illegal exportation of GBL from Poland, there also exist a number of relatively straight-forward synthetic routes utilizing less-regulated precursors. GHB is illicitly used for a variety of purposes including relaxation, improved sleep, enhanced sexual stimulation (Chemsex), and as a mean of coming down from a stimulant-fueled drug binge. In club settings, it is used to enhance the experience of the music or more nefariously as a date rape drug due to its sedative effects, which are especially potent when combined with alcohol.22 There is also a small subset of the bodybuilding community that uses GHB as a supplement with the belief that it can release growth hormone and improve sleep when cycling testosterone or other steroids.23 Hollywood stars employ GHB to ‘lean out’ body mass.24 It is commonly referred to by its street names “Georgia Home Boy”, “Vita G”, “Cherry Meth”, “Grievous Bodily Harm”, 4 ACS Paragon Plus Environment

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“Scoop”, “Fantasy”, “Liquid Ecstasy”, “Liquid X”, “Goop”, “Easy Lay”, “Blue Nitro”, “OxySleep”, “G-riffick”, “Wolfies”, “Fantasy”, “Jib”, “Soap”, “Gamma G”, or simply “G”.1 Typically sold in liquid form by the capful, or “swig”, GHB costs between $5 to $25 dollars per capful, and users claim GHB provides all the “euphoria of alcohol without the sloppy side effects and slurred speech.”24 However, GHB causes a number of short-term (aggressive behavior, amnesia, confusion, coma, death, depression, headache, seizures, sweating, slowed heart rate, amongst others) and long-term side effects (amnesia, cardiovascular collapse, extreme anxiety, hallucinations, respiratory depression). Moreover, when used repeatedly, GHB causes dependence and leads to severe withdrawal symptoms including tremors, insomnia and anxiety.25 Chemical Properties and Synthesis By molecular weight GHB (1) is the smallest Schedule 1 drug, with a molecular weight of 104.1 g/mole, or 126.09 g/mole for the sodium salt (3).26 It possesses a polar surface area of 57.53 Å2 and a ClogP -0.581.27 Its carboxylic acid has a pKa of 4.672 while its terminal hydroxyl group has a pka of 14.934.28 It is described as having a slightly salty taste, though this can be difficult to detect when combined with alcohol. It is a clear, colorless liquid with the appearance of water, and infinitely soluble in water, ethanol and many other solvents, hence the ease in ‘spiking drinks’ undetected. GHB can be administered orally (88%F), via injection or through inhalation. Once consumed orally, the onset of action occurs within 5-25 minutes, and can be fully cleared in 4-6 hours (t1/2 ~30-60 minutes).23 The absence of a chromophore, coupled with rapid degradation renders GHB hard to detect in plasma post a ‘date rape’ incident.25

Scheme 1. First documented syntheses of GBL and GHB by Zaitsev in 1874.29,30 O Cl Cl

Na(Hg), Et2O AcOH

O O

O 9

NaOH Ba(OH)2

Na+

H 2O 80°C

O O

OH

+

O

Ba2+ O

OH

2

GHB (1) Salts

4 (GBL)

The first reported synthesis of GHB was conducted by Aleksandr Zaitsev in 1874.29 He reported the formation of GBL (4) via the reductive cyclization of succinyl chloride using sodium amalgam (Scheme 1). GBL (4) was found to hydrolyze in the presence of barium and sodium hydroxide to form the respective GHB salts.30 The synthesis of GHB has largely remained the same, though the initial cyclization has been eliminated in favor of beginning the process with 4 directly.



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Scheme 2. Synthesis of sodium oxybate (3, XyremTM) employed by Jazz Pharmaceuticals.2,31 O

Ca2+

O

OH

O

O NaOH, Ca(OH)2, Mg(OH)2 H 2O

Na+

OH

O

80°C 4 (GBL)

2

O

O

Mg2+

OH

O

2

GHB (1) Salts

The synthesis for the commercially licensed Xyrem™ proceeds via the previously described base-enabled hydrolysis of GBL (4).2 GHB is clinically administered as the sodium salt, sodium oxybate, 3, though formulations involving other salts have also been patented (Scheme 2).31 These salts are used to enable formulation as a powder rather than an aqueous solution, by reducing the hygroscopicity of the final product. The recent listing of GBL (4) as a schedule 1 substance necessitates that clandestine chemists synthesize GBL themselves prior to producing GHB.

Scheme 3. BASF cyclization. Procedure employed by BASF for industrial-scale synthesis of GBL.32 1) CO (g) +

(g) 2)

Rh(CO)2acac Et3N/H2O 240 atm

O

H2, 200°C 197 atm

O

1,4-Dioxane 10

11

NaOH H 2O 80°C

4 (GBL)

Na+

O OH

O 3

One synthetic route amenable to large scale production involves GBL formation via the rhodium-catalyzed cyclization of carbon monoxide (10) and acetylene (11) in a high pressure batch reactor (Scheme 3). This produces a mixture of GBL (4) and 2(5H)furanone, which can be reduced in situ through the high pressure addition of hydrogen gas. This process is currently employed by BASF and has been shown to be high-yielding (86%) on an industrial scale.32 GHB’s salt forms, such as 3, are then accessible via the familiar base-enabled hydrolysis. Though this route shows commercial viability on a large scale, it is impractical to most chemists due to the high pressures required for the initial cyclization. The rhodium catalyst employed is also relatively difficult to obtain, and could prove problematic for most forms of home-grown purification. 6 ACS Paragon Plus Environment

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Scheme 4: THF-based Synthesis. GBL formation via THF oxidation.33

O O

12 (THF)

Ca(OCl)2 MeCN

NaOH O

Na+

H 2O 80°C

4 (GBL)

O OH

O

3

Perhaps the most practical synthetic route involves the production of GBL (4) via the oxidation of tetrahydrofuran (12, THF) by calcium hypochlorite, which is readily available in household bleach products.33 This oxidation has also been reported using various peroxides, and transition-metal complexes as oxidizing agents.34-36 Given THF’s ubiquity as a common organic solvent, it is much more easily obtained than GBL (4), and thus offers a much less regulated approach to the synthesis of GHB (1) and related salt forms. Based on these synthetic schemes, readily employed by clandestine chemists, GBL (4) is likely a common “impurity” in GBH preparations. Busardò found that out of 30 illicit preparations of street “G”, all had significant quantities of GBL (mean concentration of 760.7 ± 91.46 mg/mL), whereas only 27/30 possessed GHB (and at very low concentrations: 1.60 mg/mL ± 0.88).37 Behavioral and Molecular Pharmacology One concerning trait of GHB’s dose profile is the narrow range between effective and lethal doses (Table 2). Table 2. Behavioral pharmacology by plasma concentration. Effects measured for naïve users. Highlighted region indicates desired pharmacological effects.9,23 Behavioral Pharmacology by Concentration Plasma Concentration(mg/L) Response 0-63 Awake 63-99 Awake or light sleep 100-150 Slurred speech, impaired movement or light sleep 151-244 Medium sleep 244-395 Deep sleep/Coma 395-500 Coma >500 Death

Given that the desired effects are usually achieved at plasma concentrations of 60-100 mg/L, and overdose is achieved at 500 mg/L, GHB possesses a therapeutic index (TI) of 7 ACS Paragon Plus Environment


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only 8.23 For context, the illicit drug cocaine38 has a TI of 15, and the only Schedule 1 drug with a lower value than GHB is heroin39 at 6.40 This narrow safety margin is the primary danger of GHB as a drug of abuse. As GHB is often ingested with alcohol or other drugs of abuse, the TI is further reduced as a depressant. While GHB’s behavioral profile appears to be depressive in nature, the drug has also been shown to elicit stimulant-like responses at lower concentrations.41,42 This seemingly paradoxical mix of drug interactions is due to GHB’s ability to engage both GABAB and GHB-type receptors, depending on its concentration in the brain.4,43,44 The mechanism of action for this biphasic behavior is not fully understood. Current research into GHB’s pharmacology relies on series of tool compounds for each receptor (Figure 2). These studies indicate that GHB’s sedative and euphoric effects, as well as its abuse liability are primarily due to its interaction as a weak agonist at the GABAB receptor (EC50 ≈ 100 µM).45 Its excitatory effects have been linked to its highaffinity binding to the GHB receptor GPR172A (EC50 = 130 nM).46 GABAB agonists have long been of interest in neuroscience due to their applicability in the clinical treatment of muscle spasms and severe alcoholism. (R)-Baclofen (13) is of particular importance, as it serves as a selective GABAB agonist with an EC50 of 32 nM, and has been used as a muscle relaxant under the brand names Lioresal™ and Gablofen™ since the early 1960s.47-49 SKF-97541 (14) is another relevant GABAB agonist, as it has been shown to actively diminish reward-seeking behavior in mice, particularly in the context of ethanol self-administration.50 This may provide some support to the therapeutic viability of GABAB agonism as a means of treating chronic alcoholism, though further research suggests that such ligands potentiate the same sedative effects as ethanol and simply serve as replacement drugs, rather than actively suppressing cravings.12,51,52

OH O

H 2N

H 2N

O P OH

Cl H 2N 13 (R)-Baclofen GABAB agonist

14 SKF-97541 GABAB agonist

O O P OH O

HO

15

O

OH

16

CGP-35348 GABAB antagonist

OH

HO

O

T-HCA GHB receptor agonist

17 NCS-382 GHB receptor antagonist

Figure 2. GABAB (13-15) and GHB receptor (16,17) tool compounds.

GHB has been shown to exhibit a more moderate abuse liability than that of other Schedule 1 drugs such as phencyclidine (PCP)53 and cocaine,38 which is likely due to its activity as both a facilitator of dopamine release, as well as a CNS depressant.52,54 Further 8 ACS Paragon Plus Environment

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studies have demonstrated its abuse potential to be roughly equal to that of ethanol.37 It has been shown to be self-administered in baboons, and to produce dependence in humans at a threshold of 3-6 daily uses.55,56 This dependence is particularly worrying given the drug’s narrow dose-safety margin. The pharmacology of GHB’s abuse liability has for the most part remained elusive, though some recent studies have indicated the GABAB receptor as at least partially responsible. This idea is supported by the reportedly similar addictive nature of baclofen, given that both substances act as GABAB agonists, though baclofen does so selectively.57 Further studies have demonstrated GABAB’s role in the reward process, and this offers a plausible explanation for why these GABAB agonists elicit similar addiction profiles.58 Though this clarifies the sedative nature of GHB, it offers little explanation for its excitatory effects, let alone its natural role in the CNS. It was not until the discovery of a GHBspecific receptor in 1982 that GHB’s concentration-dependent nature was fully understood.59 Through competitive binding experiments, Benavides et al. were able to show binding of GHB to a receptor independent of the GABAergic system, and that it was not displaced from this receptor in the presence of the several-fold more potent GABAB agonist baclofen (13).59 This result gave credence to the existence of a GHB-specific receptor in the rodent CNS. This idea was supported through further research utilizing the selective GABAB antagonist GCP-35348 (15). By pre-dosing mice with this antagonist before administering known GABAB agonists GHB and baclofen, Nissbrandt and colleagues found a distinct drop-off in the depressive symptoms tied to GABAB agonism. This result further confirmed the hypothesis that these effects are due to GHB’s interaction as a weak GABAB agonist, rather than its interactions with the GHB receptor.60 Findings like these spurred the search for a selective GHB-receptor agonist to more directly study the effects of this receptor. Trans-4-hydroxycrotonic acid (16, T-HCA) was found to be such a compound with a 4-fold greater affinity for the GHB receptor than GHB itself.61 By administering this selective GHB-receptor agonist to rats, Castelli et al. observed increased levels of extracellular glutamate, and a complete absence of the depressive symptoms associated with GHB, suggesting the excitatory nature of the GHB receptor.62 This excitatory release of extracellular glutamate is thought to be the cause of GHB-induced seizures at higher doses.42 One final analogue that has been used in the study of GHB’s pharmacology is NCS-382 (17). This potent GHB receptor antagonist (IC50 = 42-200 nM) has been shown to reduce catalepsy in rats that had previously been dosed with GHB.63,64 It shows therapeutic potential as a treatment for both GHB overdose and succinic semialdehyde 9 ACS Paragon Plus Environment


dehydrogenase deficiency (SSADHD) in humans, though it has never been developed for clinical use due to the fear of off-target interactions with the GABAB receptor.62,65 To date, no studies have been conducted utilizing a totally selective GHB receptor antagonist, though the therapeutic value seems evident. Such a drug could also serve as an important tool in elucidating the role of endogenously-produced GHB, which remains unclear despite its prevalence in the human brain at concentrations ranging from 0.5 – 1.0 mg/L.66 These interactions help to explain the concentration-dependent effects of GHB. At lower concentration, high affinity binding to the GHB receptor dominates, resulting in the release of extracellular glutamate and excitatory symptoms. In contrast, higher concentrations of GHB shunt pharmacology towards weak agonism at the GABAB receptor, resulting in a depressive response through the inhibition of neurotransmitter release. These divergent pharmacological responses based on GHB leads to the phenomenon known as “rebounding”, wherein users, in the midst of GHB-induced sleep, experience sudden restlessness. Drug Metabolism and Pharmacokinetics Unlike most elicit substances, the metabolism of GHB produces no active metabolites. Due to its structural similarity to GABA, and by extension the primary amino acid glutamate, GHB is readily converted into a host of simple metabolites that are wellintegrated into the body’s everyday function. Its 2-step conversion into succinic acid allows for total elimination of the drug from the body through the citric acid cycle, leaving behind only carbon dioxide and water.67 This process takes place rather quickly with GHB exhibiting an in vivo half-life ranging from 30 to 50 minutes, with a Cmax of 63 mg/mL occurring 40 minutes after ingestion of a 3 gram dose.68,69 Ingestion of common GHB prodrugs such as gamma-butyrolactone (4, GBL) and 1,4-butanediol (5, BD) leads to a spike in GHB plasma levels due to their rapid conversion into the drug itself (t1/2 ~ 1 minute).9,70 This quick metabolism is partially responsible for the popularity of these drugs as GHB substitutes. This is particularly evident given that GBL (4) possesses a higher oral bioavailability than that of GHB.9 As such, GBL (4) leads to a much faster onset of its depressive effects. In a 32 mg/kg intragastric dose scheme, BD (5) leads to a GHB (1) Cmax of 58.5-152 µmol/L, while GBL (4) leads to a GHB (1) Cmax of 15-588 µmol/L.11,55 It seems counterintuitive that a polar, negatively charged species accesses the CNS and crosses the blood brain barrier. Clearly, GHB must be subject to a carrier-mediated transport mechanism, and it has been shown that GHB crosses the blood-brain-barrier via the monocarboxylate transporter (MCT1).71


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Commmon Prodrugs

O

HO

O

OH 5 (BD)

4 (GBL)

O

Alcohol dehydrogenase

HO

OH NH2

O Lactonase

HO

20 glutamic acid

H 18 gamma-hydroxybutyraldehyde

glutamic acid decarboxylase

Aldehyde dehydrogenase O HO

OH gamma-hydroxybutyric acid (1, GHB)

O

GHB dehydrogenase succinic semialdehyde reductase

O O

OH

O

GABA transaminase

19 succinic semialdehyde

H 2N

OH gamma-aminobutyric acid (2, GABA)

Succinic semialdehyde dehydrogenase

Succinic acid Citric Acid Cycle

Figure 3. Metabolic pathway of GHB (1), GBL (4) and BD (5).

GBL is converted directly to GHB by the oxidative activity of serum lactonases (Figure 3). BD is successively oxidized into gamma-hydroxybutyraldehyde by alcohol dehydrogenase, and GHB by aldehyde dehydrogenase.47 Once GHB reaches sufficient concentration in the body, it is reduced to succinic semialdehyde (SSA) by GHB dehydrogenase.72 SSA can then either be aminated into GABA through the reversible activity of GABA transaminase, or it can be oxidized by succinic semialdehyde dehydrogenase to form succinic acid, which enters directly into the citric acid cycle and ultimately leads to elimination of the drug.73 This enzyme is crucial in eliminating GHB from the body whether it be produced endogenously or taken exogenously. A rare genetic deficiency in this enzyme (SSADHD) prevents the individual from being able to effectively eliminate GHB from the brain, as it is continually produced through the reduction of SSA by succinic semialdehyde reductase (SSR).74 This leads to a host of symptoms that directly mirror the adverse effects of GHB.75-77 The reversible activity of GABA transaminase was once thought to be important in the pharmacological effects of GHB. It was postulated that it offered the ability to produce GABA from exogenous GHB, thereby activating GABAA receptors to produce its inhibitory effects.78-80 Though this hypothesis is supported by a series of in vitro studies, it is contradicted by more recent in vivo studies which demonstrate the leftward preference of 11 ACS Paragon Plus Environment


the GABA transaminase equilibrium in Figure 2. That is, these studies reveal the preferential role of GABA transaminase as a means of eliminating excess GABA by converting it into SSA, rather than as a means of synthesizing GABA from SSA.7,81 Adverse Effects and Societal Impact GHB is commonly used for its euphoria-inducing effects including relaxation, decreased inhibition, and enhanced sexual stimulation (Chemsex), among others. The aphrodisiacal effects reported by users is thought to be the combined result of decreased inhibition and sedation, rather than a direct increase in sexual arousal.82 The sedative nature of GHB allows the user to rest whether due to insomnia or coming down from a cocktail of drugs, most commonly including MDMA “molly”.83 In larger quantities, GHB can induce a nearunarousable unconscious state.84 This can be exacerbated when combined with other drugs or alcohol. GHB’s adverse effects include vomiting, CNS depression, bradychardia, seizures, and death, often due to respiratory failure.23 GHB overdose is not difficult to achieve due to confusion over appropriate dosing amounts, especially in the context of GHB-doped solutions. This is particularly worrying given that each batch produced by home chemists can be vastly different in terms of chemical composition, due to the tendency of such suppliers to sometimes sell a GBL/GHB mixture.85 compounding the dangers associated with GHB is a general lack of how to treat overdose when it occurs. There is no known cure for GHB overdose, and often times users are slow to seek medical attention, if at all, instead opting to “sleep-off” the effects of the drug.83 Some chronic users have reported GHB withdrawal which most commonly manifests as tremors, anxiety, depression, insomnia, and in rare instances, seizures. Withdrawal usually occurs as the result of sudden cessation after habitual use and is thought to be linked to the depressive nature of the drug’s GABAB agonism.23 A sudden drop-off in this interaction leads to a spike in extracellular glutamate, often resulting in these common withdrawal symptoms.42 A rising problem with GHB is its use as a date rape drug, due to its high solubility in both ethanol and water, combined with its colorless appearance and lack of a distinct odor. This is compounded by its short half-life and natural presence in the body which makes it very difficult to identify its use in date rape allegations. Less than 5% of the initial dose is often recoverable in the user’s urine, with a detection window of only 3-10 hours after administration.9 In the context of date rape, GHB is often unknowingly dissolved in the victim’s drink.86 Given that the metabolites of GHB are nothing more than water and carbon dioxide, it becomes exceedingly difficult to detect once ingested. This difficulty of 12 ACS Paragon Plus Environment

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detection is further exacerbated by GHB’s short half-life. Victims of GHB-mediated daterape are often encouraged to get tested within 6 hours of sexual assault in order to track the drug before it is metabolized such that its plasma concentration falls below its normally-observed endogenous levels.9,87 Some techniques have been developed to circumvent this rapid metabolism by utilizing samples other than blood and urine for forensic detection of exogenous GHB. Hair samples provide the unique opportunity to analyze GHB levels several weeks after administration, while fingernails seem to provide a reliable baseline for endogenous GHB concentrations.88-90 If used in conjunction, these methods could prove valuable in GHB-mediated sexual assault cases even weeks after the fact. In one study of sexual assault victims, GHB was found to be 5 times more prevalent than flunitrazepam (Rohypnol/“Roofies”), the most commonly referenced daterape drug in popular culture.23 The discrepancy in legal statuses of GHB and GBL has quickly become an imminent political issue is some countries. Given the availability of the prodrug GBL in these areas, the effective access to GHB has scarcely decreased, if at all, despite its legal restriction. GBL’s rapid metabolism into GHB promotes the same effects at equimolar doses, and considering its higher oral bioavailability increases its effective potency to roughly 3 fold that of GHB.91 GBL is often taken with an alcoholic drink due to its harshness on the esophagus and stomach, further obfuscating exact dosage. As such, a GBL-doped drink could prove fatal based on the ambiguity of dosage, even for a user that has built up a tolerance through habitual use of GHB. Both GBL and GHB are most commonly encountered as colorless solutions and as such it is very easy for a user to take an inaccurate dose over confusion as to which drug they are taking. The two are described to remain slightly different in taste, but when mixed with a drink this may be harder to distinguish.18,23 Moreover, as the majority of illicit GHB preparations contain predominantly GBL, this is a significant concern. GHB has been shown to have negative effects on the body when used in conjunction with other substances, most notably alcohol. When used together, GHB can elicit synergistic cooperativity between the depressive effects of each substance, causing the user to become unconscious.92,93 This allows for the individual to easily be taken advantage of, as this comatose state may be mistakenly interpreted as mere inebriation by other clubgoers. This demonstrates GHB’s role in fueling sexual assault within the club scene whether the drug be given surreptitiously, or taken willingly. GHB was once a popular bodybuilding supplement, though its use within this community has diminished in recent years.1,23 The dosing of GHB is unclear within the bodybuilding scene, while its level of efficacy is still challenged to this day. The belief that GHB can



increase growth hormone in the body seems suspect at best however some studies suggest that there may be some truth to this idea.94 Both GHB and GBL have had major impact and garnered significant attention in the popular press and in pop culture. As of 2017, GHB was making a comeback amongst fitness addicts, and Hollywood stars were employing GHB to ‘lean out’ for new roles.24 The latter was the case when John Stamos was stopped by police in Beverly Hills, issued a DUI, and found that he was under the influence of GHB – not alcohol. According to Steven Fowkes, a chemist and executive director of the Cognitive Enhancement Research Institute, “one gram of GHB can be like a can of beer, two grams can be like three cans, and three grams can be like a case.”24 The most famous GHB binge leading to a DUI was that of actor Nick Nolte in 2002, and his now infamous mug shot (https://people.com/books/nick-nolte-2002-mug-shot-new-memoir/), which has become the face of GHB abuse – shortly after been named Sexiest Man Alive. GHB made world-wide headlines in 2007, when Australia announced a nationwide ban on ~1 million Chinesemade bead toys (Bindeez, Australia’s 2007 toys of the year) after investigations showed non-toxic 1,5-pentanediol was replaced with 1,4-butanediol (BD, 5) in the manufacturing process. Upon ingestion, BD metabolizes to the date-rape drug GHB.95 Unfortunately, several children ingested the BD-tainted beads, and subsequently suffered seizures.

History and Importance in Neuroscience Therapeutic interest in GHB began with Laborit’s characterization of its depressive pharmacological effects in 1964, though the first work within the GHB chemotype occurred in 1874 (Figure 4).3 Though popularization of his work, GHB became a common anesthetic throughout the 1960’s though it was later shelved as a result of its poor analgesic effects, and concerns over its promotion of absence seizures.96 Nevertheless, this popularity in the medical community helped motivate much of the work that ultimately led to its use in the clinical treatment of narcolepsy. Xyrem™ has been a treatment since its approval in 2002, and boasted $1.4 billion in total revenue in 2018 alone.97


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Figure 4. Timeline of GHB from discovery to a drug of abuse to a new treatment for narcolepsy.

Though the therapeutic value of GHB itself remains dubious outside of narcolepsy, its discovery and characterization has laid the groundwork for the potential treatment of myriad conditions relying on GABAergic processes, including multiple sclerosis, SSADHD, alcohol dependence, and epilepsy.98 Furthermore, its biphasic nature of dopamine release has shed much-needed-light on the role of GABA in the CNS of higher mammals. GHB holds the distinct honor of being one of only two Schedule 1 drugs to be endogenously present in the human CNS (the other being Dimethyltryptamine or DMT).99,100 Its natural abundance in the CNS of higher mammals was first reported in the 1970s, and helped drive research into its role within the GABAergic framework.101 Its wellstudied interactions with both GABAB and GHB receptors have paved the way for the discovery of many selective agonists and antagonists at these sites, allowing researchers a glance into the inner workings of the chief inhibitory system of the human CNS. Such insight is of particular importance in the context of alcohol abuse, given ethanol’s similar effects on the GABAergic system.102 GHB grew to prominence in the public eye in the 1980’s due to claims that it stimulated muscle growth, and could be used as a fat burner when combined with other dietary supplements. Its wide-spread commercial availability at this time is what motivated its subsequent popularity in the club scene. Despite its simple structure, GHB and the prodrugs thereof have had a substantial impact on neuroscience and society at large. 15 ACS Paragon Plus Environment


GHB’s complex interactions with both the GABAB and GHB receptors have played an incredibly important role in the study of the GABAergic system, and have aided in the clinical treatment of several debilitating conditions such as narcolepsy and severe alcohol abuse.103,104 They have had pronounced effects on numerous subsects of popular culture with bodybuilders, club-goers, and home drug enthusiasts alike showing interest for one reason or another. Its natural abundance in the CNS has puzzled researchers for decades, and serves as a humbling reminder that for as much as we learn in neuroscience, there always remain more mysteries to uncover. Considering all this, it’s no small wonder that GHB is nothing short of a classic molecule on the dark side of chemical neuroscience.

Author Information Corresponding Author *Email: [email protected] ORCID Craig W. Lindsley: 0000-0003-0168-1445 Author Contributions T.A.T., R.A.C. and C.W.L. all researched and wrote sections of this review manuscript. Notes The authors declare no competing financial interest. Acknowledgements We thank William K. Warren, Jr., and the William K. Warren Foundation who funded the William K. Warren Jr. Chair in Medicine (to C.W.L.). We also thank Aaron T. Garrison and Aaron M. Bender for their help in the review process.

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DARK Classics in Chemical Neuroscience: Gamma-Hydroxybutyrate

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