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`1521-0111/12/8202-226–235$25.00
`MOLECULAR PHARMACOLOGY
`Copyright © 2012 The American Society for Pharmacology and Experimental Therapeutics
`Mol Pharmacol 82:226–235, 2012
`
`Vol. 82, No. 2
`78154/3781922
`
`␥-Hydroxybutyrate (GHB)-Induced Respiratory Depression:
`Combined Receptor-Transporter Inhibition Therapy for
`Treatment in GHB Overdose
`
`Bridget L. Morse, Nisha Vijay, and Marilyn E. Morris
`Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, University at Buffalo, State
`University of New York, Buffalo, New York
`Received February 7, 2012; accepted May 4, 2012
`
`ABSTRACT
`Overdose of ␥-hydroxybutyrate (GHB) frequently causes respi-
`ratory depression, occasionally resulting in death; however,
`little is known about the dose-response relationship or effects
`of potential overdose treatment strategies on GHB-induced
`respiratory depression.
`In these studies, the parameters of
`respiratory rate, tidal volume, and minute volume were mea-
`sured using whole-body plethysmography in rats administered
`GHB. Intravenous doses of 200, 600, and 1500 mg/kg were
`administered to assess the dose-dependent effects of GHB on
`respiration.To determine the receptors involved in GHB-induced
`respiratory depression, a specific GABAB receptor antagonist,
`(2S)-(⫹)-5,5-dimethyl-2-morpholineacetic acid (SCH50911), and a
`specific GABAA receptor antagonist, bicuculline, were adminis-
`tered before GHB. The potential therapeutic strategies of receptor
`inhibition and monocarboxylate transporter (MCT) inhibition were
`assessed by inhibitor administration 5 min after GHB. The primary
`
`effect of GHB on respiration was a dose-dependent decrease in
`respiratory rate, accompanied by an increase in tidal volume,
`resulting in little change in minute volume. Pretreatment with 150
`mg/kg SCH50911 completely prevented the decrease in respira-
`tory rate, indicating agonism at GABAB receptors to be primarily
`responsible for GHB-induced respiratory depression. Administra-
`tion of 50 mg/kg SCH50911 after GHB completely reversed the
`decrease in respiratory rate; lower doses had partial effects. Ad-
`ministration of the MCT inhibitor L-lactate increased GHB renal
`and total clearance, also improving respiratory rate. Administra-
`tion of 5 mg/kg SCH50911 plus L-lactate further improved respi-
`ratory rate compared with the same dose of either agent alone,
`indicating that GABAB and MCT inhibitors, alone and in combina-
`tion, represent potential treatment options for GHB-induced re-
`spiratory depression.
`
`Introduction
`␥-Hydroxybutyate (GHB) is a short-chain fatty acid pres-
`ent endogenously in many human tissues, resulting from
`production via GABA metabolism (Maitre, 1997). GHB has
`also recently been identified as a useful therapeutic agent for
`the treatment of narcolepsy and excessive daytime sleepiness
`in the form of sodium oxybate (Xyrem; Jazz Pharmaceuticals,
`Palo Alto, CA). However, GHB has become more popularly
`
`This work was supported by the National Institutes of Health National
`Institute on Drug Abuse [Grant DA023223] and Pfizer Global Research and
`Development.
`This work was previously presented in part as an abstract: Morse B,
`Uhlander J, and Morris M. Respiratory depression in ␥-hydroxybutyrate over-
`dose: interaction with ethanol and treatment using monocarboxylate trans-
`porter inhibition, AAPS Annual Meeting and Exposition; 2011 Oct 23–27; Wash-
`ington DC. American Association of Pharmaceutical Sciences, Arlington, VA.
`Article, publication date, and citation information can be found at
`http://molpharm.aspetjournals.org.
`http://dx.doi.org/10.1124/mol.112.078154.
`
`known as a drug of abuse. According to reports from the Drug
`Abuse Warning Network, there have consistently been 1000
`to 2000 GHB-related emergency department visits reported
`annually in the United States over the past several years
`(Substance Abuse and Mental Health Services Administra-
`tion, 2011). GHB overdose can result in manifestations in-
`cluding sedation, coma, hypothermia, bradycardia, respira-
`tory depression, and death (Li et al., 1998; Sporer et al., 2003;
`Caldicott et al., 2004; Galicia et al., 2011). In a recent report
`of known GHB-associated fatalities, the most common cause
`of mortality was cardiorespiratory arrest (Zvosec et al.,
`2011). Respiratory depression with the need for mechanical
`ventilation is also frequently reported in nonfatal cases of
`GHB intoxication (Li et al., 1998; Mason and Kerns, 2002;
`Liechti and Kupferschmidt, 2004).
`Although respiratory depression is a common symptom of
`GHB overdose, neither the dose-dependent effects of GHB on
`this measure nor the neurotransmitter receptors involved in
`
`ABBREVIATIONS: GHB, ␥-hydroxybutyrate; MCT, monocarboxylate transporter; SCH50911, (2S)-(⫹)-5,5-dimethyl-2-morpholineacetic acid; LC,
`liquid chromatography; MS/MS, mass spectrometry; Emax, maximum effect; Td, duration of effect; AUC, area under the plasma concentration-time
`curve; ABEC, area below the effect curve.
`
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`GHB-induced respiratory depression have been investigated.
`There are several proposed actions of GHB, including 1)
`direct action at GABAB receptors (Bernasconi et al., 1992), 2)
`direct action at its own putative GHB receptor (Maitre,
`1997), and 3) indirect action at GABA receptors via GABA
`production/release (Hechler et al., 1997; Gobaille et al.,
`1999). Although evidence exists for each of these mechanisms
`in vitro and/or in vivo, many of the toxicological effects of
`GHB, including sedation, hypothermia, and fatality, can be
`attributed to agonism at GABAB receptors (Carai et al., 2001,
`2005; Kaupmann et al., 2003).
`Along with a complex pharmacologic profile, the pharma-
`cokinetics of GHB are also notably complicated. In humans,
`GHB exhibits dose-dependent pharmacokinetics, even at
`therapeutic concentrations (Palatini et al., 1993). Rats simi-
`larly display nonlinear pharmacokinetics, due to several con-
`centration-dependent processes including saturable oral absorp-
`tion, saturable metabolism, and saturable renal reabsorption
`(Lettieri and Fung, 1979; Morris et al., 2005). In both humans and
`rats, GHB metabolism is the predominant route of elimination at
`low doses, and renal excretion of unchanged drug is minimal
`(Lettieri and Fung, 1976; Brenneisen et al., 2004). Although lim-
`ited information exists for supratherapeutic GHB doses in hu-
`mans, it has been well documented in rats that renal clearance
`becomes an increasingly important route of elimination as GHB
`doses are increased (Morris et al., 2005). This nonlinear renal
`clearance can be attributed to a concentration-dependent trans-
`port process, leading to saturable renal reabsorption, demon-
`strated in our laboratory to involve the group of transporters
`known as monocarboxylate transporters (MCTs) (Morris et al.,
`2005; Wang et al., 2006). MCTs are proton-dependent transporters
`expressed throughout the body, and GHB is an identified sub-
`strate for MCTs 1, 2, and 4 (Wang et al., 2006; Wang and Morris,
`2007). The ubiquitous expression of these transporters includes
`that in the intestine, kidney, and brain, regions of interest regard-
`ing GHB pharmacokinetics. Because of their role in the renal
`reabsorption of GHB, inhibition of these transporters represents a
`potential therapeutic strategy for GHB overdose. This strategy has
`been demonstrated to translate to in vivo effects on GHB disposi-
`tion, and administration of MCT inhibitors increases the renal and
`total clearance in animal models of GHB overdose (Morris et al.,
`2005; Wang et al., 2008a,b). Likewise, the administration of the
`MCT inhibitor L-lactate, in combination with osmotic diuresis,
`increases the renal clearance of GHB in humans, as demonstrated
`in our pilot clinical study (Morris et al., 2011).
`The first aim of this research was to investigate the dose-
`response relationship of GHB-induced respiratory depres-
`sion, including the primary neurotransmitter receptors in-
`volved in eliciting this effect. The second was to assess
`potential treatment strategies, including MCT and receptor
`inhibition, for improving GHB-induced respiratory depres-
`sion, because the application of these strategies for treating
`this pharmacodynamic endpoint have not been evaluated
`previously.
`
`Materials and Methods
`Chemicals and Reagents. Sodium GHB used in these studies
`was provided by the National Institute on Drug Abuse. Deuterated
`GHB (GHB-d6) was purchased from Cerilliant Corporation (Round
`Rock, TX). Sodium L-lactate and bicuculline methiodide were pur-
`chased from Sigma-Aldrich (St. Louis, MO). (2S)-(⫹)-5,5-Dimethyl-
`
`GHB-Induced Respiratory Depression
`
`227
`
`2-morpholineacetic acid (SCH50911) was purchased from Tocris Bio-
`science (Ellisville, MO). High-performance liquid chromatography-
`grade acetonitrile and acetic acid were purchased from Honeywell
`Burdick & Jackson (Muskegon, MI).
`Animals and Animal Surgery. Male Sprague-Dawley rats (Har-
`lan, Indianapolis, IN) weighing 270 to 330 g were used for all exper-
`iments. Animals were housed under controlled temperature and
`humidity with an artificial 12-h light/dark cycle, and food was avail-
`able ad libitum. All animal protocols were approved by the Institu-
`tional Animal Care and Use Committee at the University at Buffalo.
`Animals were allowed to acclimate to their environment for a mini-
`mum of 1 week before surgical implantation of jugular and femoral
`vein cannulae under anesthesia with ketamine-xylazine. Cannulae
`were flushed daily with 40 IU/ml heparinized saline to maintain
`patency. Animals were allowed a minimum of 72 h for recovery from
`surgery before drug administration.
`Plethysmography. Measurement of respiration in these studies
`was performed using a whole-body plethysmograph (model PLY4213;
`Buxco Research Systems, Wilmington, NC). Plethysmography equip-
`ment included unrestrained plethysmography chambers consisting
`of a main (animal) chamber and reference chamber for buffering
`changes in atmospheric pressure. The plethysmography chambers
`were connected to a Rodent Bias Flow Supply (BFL0250) to draw
`expired CO2 out of the chambers and provide a smoothed flow of
`room air at a flow rate of 2.5 l/min per chamber. The plethysmogra-
`phy chambers included ports to which a pressure sensor was con-
`nected and led to the MAX 1500 preamplifier. Signals were collected,
`visualized, and quantitated using BioSystem XA software. Two ad-
`ditional ports were included in the chambers for the insertion of
`jugular and femoral vein cannulae, allowing for drug administration
`and blood sampling. Urine was collected at the base of the chamber
`at intervals by opening an additional port at the base. Calibration of
`chamber pressure was performed before every experiment by injec-
`tion of 5 ml of air through the base port. At each recording, signals
`were collected for six intervals of 10 s each and averaged to represent
`1 min of recording. Measurements for the parameters of respiratory
`frequency (rate), tidal volume, and minute volume (rate 䡠 tidal vol-
`ume) were quantitated for each recording.
`Pharmacokinetic/Pharmacodynamic Studies. Rats were
`placed in plethysmography chambers 1 h before drug administration
`and allowed to acclimate to the chambers for 45 min before five
`baseline measurements of 1 min each were collected over 15 min. In
`all studies, GHB administration was considered time 0, and respi-
`ration measurements were recorded at 2.5, 5, 7.5, 10, 15, 20, 25, and
`30 min and every 15 min thereafter for 480 min. Blood samples were
`collected, and collection times were optimized for each GHB dose
`according to previous studies (Felmlee et al., 2010b, 2011). Urine was
`collected at intervals up to 480 min. For overlapping pharmacoki-
`netic/pharmacodynamic time points, blood and urine samples were
`taken directly after the recording of respiratory measurements.
`Dose-Dependent Effects of GHB on Respiration. To assess
`the dose-response relationship of GHB-induced respiratory depres-
`sion, rats were administered GHB intravenously in doses of 200, 600,
`and 1500 mg/kg (four to six animals per dose). GHB was injected over
`1 to 2 min as a 300 mg/ml solution in sterile water via the jugular
`vein cannula. A placebo control group received a 5 ml/kg saline bolus.
`Neurotransmitter Receptors Involved in GHB-Induced Re-
`spiratory Depression. To determine the primary receptors in-
`volved in GHB-induced respiratory depression, rats were pretreated
`with specific receptor antagonists. Bicuculline methiodide (5 mg/kg)
`was administered for inhibition of GABAA receptors and SCH50911
`(150 mg/kg) for inhibition of GABAB receptors (three to four animals
`per group). Inhibitors were administered immediately after the col-
`lection of baseline respiratory measurements and 1500 mg/kg GHB
`was administered 5 min later. Data from dose-dependent experi-
`ments were used as the control. Bicuculline methiodide was admin-
`istered as a 5 mg/ml solution in saline and SCH50911 as a 50 mg/ml
`solution in saline via the jugular vein cannula.
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`228
`
`Morse et al.
`
`Potential Treatment Strategies. To assess the effect of poten-
`tial treatment strategies on GHB-induced respiratory depression,
`treatments were administered intravenously 5 min after 1500 mg/kg
`GHB. Treatment strategies included SCH50911 (2.5, 5, 10, and 50
`mg/kg), the MCT inhibitor L-lactate (66 mg/kg bolus followed by a
`302.5 mg/kg/h infusion for 8 h), and combination therapy of 5 mg/kg
`SCH50911 plus the same dose of L-lactate. Treatment groups in-
`cluded three to five animals per group, and were compared with the
`1500 mg/kg control group from dose-dependent experiments to de-
`termine the effects of treatment on GHB-induced respiratory depres-
`sion. The same L-lactate dose was also administered alone at time 0
`to assess potential effects of this agent on respiration. In these
`experiments, SCH50911 was administered as a 2.5, 5, 10, or 50
`mg/ml solution in saline via the jugular vein cannula and L-lactate as
`a 40 mg/ml solution in sterile water via the femoral vein cannula.
`Plasma and Urine Sample Analysis. GHB plasma concentra-
`tions were determined using an LC-MS/MS method, similar to those
`published previously (Fung et al., 2008; Felmlee et al., 2010a).
`Plasma samples were prepared by adding 5 ␮l of GHB-d6 (125 ␮g/ml)
`to 50 ␮l of sample. Plasma standards and quality controls were
`prepared by adding 5 ␮l of GHB-d6 and 5 ␮l of GHB stock solution to
`45 ␮l of blank plasma, and 800 ␮l of 0.1% formic acid in acetonitrile
`was added to precipitate the plasma proteins. The samples were
`vortexed, followed by centrifugation at 10,000g for 20 min at 4°C.
`Then 750 ␮l of the supernatant was aspirated and evaporated under
`a stream of nitrogen gas. The samples were reconstituted in 250 ␮l of
`aqueous mobile phase.
`The LC-MS/MS assay was performed on an Agilent 1100 series
`high-performance liquid chromatography system with binary pump
`and autosampler (Agilent Technologies, Santa Clara, CA) connected
`to a PerkinElmer Sciex API 3000 triple quadrupole tandem mass
`spectrometer with a TurboIonSpray (Applied Biosystems, Foster
`City, CA). Chromatographic separation was achieved by injecting 7
`␮l of sample on an Xterra MS C18 column (250 ⫻ 2.1 mm i.d., 5-␮m
`particle size; Waters, Milford, MA). Mobile phase A consisted of 5:95
`acetonitrile-water with 0.1% acetic acid and mobile phase B con-
`sisted of 95:5 acetonitrile-water with 0.1% acetic acid. The flow rate
`was 200 ␮l/min with the following gradient elution profile: 100 to
`68% A over 7 min; 68 to 10% A over 3 min; and 10 to 100% over 5 min
`for a total run time of 15 min. The mass spectrometer was operated
`in a positive ionization mode with multiple reaction monitoring.
`Q1/Q3 m/z ratios for the parent/product ions of GHB and GHB-d6
`were 105.2/87.2 and 111.1/93.2, respectively. The mass spectrometer
`parameters were optimized at a declustering potential of 18 V, fo-
`cusing potential of 100 V, collision energy of 20 V, entrance potential
`of 10 V, and collision cell exit potential of 5 V. The ion spray voltage
`was set at 5500 V with temperature at 350°C. Nebulizer and curtain
`gas flow were set at 10 and 8 ml/min, respectively. The retention
`time for GHB was 4.15 min. The data were analyzed using Analyst
`software version 1.4.2 (Applied Biosystems).
`Regression analysis of peak area ratios of GHB/GHB-d6 to GHB
`concentrations was used to assess linearity of the curve. The intra-
`day and interday precision and accuracy were determined using
`quality control (QC) samples at 10 ␮g/ml (low QC), 125 ␮g/ml (me-
`dium QC), and 400 ␮g/ml (high QC). For determination of the intra-
`day precision and accuracy, quality control samples were analyzed in
`triplicate on each day, whereas for the interday precision and accu-
`racy, quality control samples were analyzed on three different days.
`A calibration curve was run on each analysis day along with the
`quality controls. The precision was determined by the coefficient of
`variation, and accuracy was measured by comparing the calculated
`concentration with the known concentration.
`Urine samples were prepared and analyzed for GHB using a
`previously described LC-MS/MS method (Felmlee et al., 2010b).
`Plasma lactate concentrations were determined using a YSI 1500
`Sport Lactate Analyzer (YSI, Inc., Yellow Springs, OH).
`Data and Statistical Analysis. Pharmacokinetic parameters
`were determined via noncompartmental analysis using WinNonlin
`
`5.2 (Pharsight, Mountain View, CA). The area below the plasma
`concentration-time curve (AUC) was determined using the trapezoi-
`dal method. Total clearance (Cl) was determined as dose/AUC. Renal
`clearance (ClR) was determined as Ae/AUC, where Ae represents the
`amount excreted in the urine. Percentage of urinary excretion was
`calculated as Ae/dose. Metabolic or nonrenal clearance (Clm) was
`calculated as Cl ⫺ ClR. The pharmacodynamic descriptors of area
`below the effect curve (ABEC), maximum effect (Emax), time of max-
`imum effect (Tmax), and duration of effect (Td) were used to deter-
`mine the effects of inhibitor administration on GHB-induced respi-
`ratory depression. ABEC was calculated using WinNonlin. Td was
`determined for each animal as the time to return to its individual
`baseline respiratory frequency. Statistical analysis was performed
`using SigmaPlot 10.0 (Systat Software, Inc., San Jose, CA). Differ-
`ences were considered significant when p ⬍ 0.05. One-way analysis
`of variance followed by Dunnett’s or Tukey’s post hoc tests was used
`to determine statistically significant differences in mean pharmaco-
`kinetic and pharmacodynamic parameters between groups. Paired t
`tests were used to determine statistically significant changes in
`respiratory parameters compared with baseline. In determining the
`effects of L-lactate alone on respiration, the average of the last hour
`of respiratory measurements was compared with the individual av-
`erage baseline values. Mean steady-state lactate plasma concentra-
`tions were calculated as the average of hourly values beginning at 60
`min.
`
`Results
`Plasma GHB LC-MS/MS Assay. The lower limit of
`quantification for GHB in plasma was found to be 5 ␮g/ml
`with acceptable error in precision and accuracy of less than
`20%. The endogenous concentrations of GHB in plasma are
`negligible compared with GHB concentrations obtained
`after administration of the lowest dose in our studies
`(Fung et al., 2004); therefore, the endogenous concentra-
`tions were not included in the calculation of GHB concen-
`trations in plasma. The standard curve for GHB ranged
`from 5 to 500 ␮g/ml based on regression analysis of peak
`area ratios of GHB/GHB-d6 to GHB concentrations with a
`correlation coefficient (r2 ⬎0.999). The intraday and inter-
`
`TABLE 1
`Intraday and interday accuracy and precision for GHB in rat plasma
`Each measured concentration is the mean of triplicate measurements. The analysis
`was performed over 3 days.
`
`Nominal
`Concentration
`
`Measured
`Concentration
`
`S.D.
`
`Precision
`
`Accuracy
`
`Intraday
`
`Interday
`
`␮g/ml
`
`10.8
`121
`375
`10.5
`118
`368
`
`0.12
`4.36
`6.93
`0.32
`2.96
`6.38
`
`CV%
`1.07
`3.60
`1.85
`3.05
`2.50
`1.73
`
`%
`107.7
`96.8
`93.7
`105.2
`94.9
`92.0
`
`10
`125
`400
`10
`125
`400
`
`CV, coefficient of variation.
`
`TABLE 2
`Nonlinear pharmacokinetics of GHB
`GHB was administered intravenously. Data are presented as mean (S.D.); n ⫽ 4 to
`6. One-way analysis of variance followed by Tukey’s post hoc test was used to
`determine statistically significant differences in pharmacokinetic parameters.
`
`200 mg/kg
`
`600 mg/kg
`
`1500 mg/kg
`
`Cl, ml 䡠 kg⫺1 䡠 min⫺1
`6.00 (0.74)a
`7.60 (0.29)
`ClR, ml䡠 kg⫺1 䡠 min⫺1
`1.68 (0.75)a
`0.444 (0.20)
`26.7 (11)a
`6.0 (3)
`Urinary excretion, %
`a Significantly different from 200 mg/kg GHB (P ⬍ 0.05).
`b Significantly different from 200 and 600 mg/kg GHB (P ⬍ 0.05).
`
`5.16 (0.70)a
`3.18 (0.66)a,b
`60.1 (7)a,b
`
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`Fig. 1. Dose-dependent effects of GHB
`on measures of respiration. GHB was
`administered intravenously at time 0.
`Data are presented as mean ⫾ S.D.;
`n ⫽ 4 to 6.
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`Fig. 2. Effect of GHB administration
`on respiratory pattern. Displayed are
`sample 10-s interval plethysmogra-
`phy traces obtained at baseline (A)
`and 30 min after administration of
`GHB 1500 mg/kg i.v. (B).
`
`TABLE 3
`Effects of specific receptor antagonists on the
`pharmacokinetics/pharmacodynamics of GHB (1500 mg/kg i.v.)
`SCH50911 (150 mg/kg) and bicuculline methiodide (5 mg/kg) were administered
`intravenously 5 min before GHB. Data are presented as mean (S.D.); n ⫽ 3 to 5.
`One-way analysis of variance followed by Dunnett’s post hoc test was used to
`determine statistically significant differences in mean pharmacokinetic and phar-
`macodynamic parameters with inhibitor administration compared with those with
`GHB alone.
`
`GHB
`
`GHB ⫹
`SCH50911
`
`GHB ⫹ Bicuc-
`ulline
`
`5.16 (0.70)
`3.18 (0.66)
`1.99 (0.17)
`10,500 (2700)
`
`6.07 (0.47)
`2.84 (0.32)
`3.23 (0.78)*
`—a
`
`5.02 (0.14)
`2.72 (0.78)
`2.30 (0.63)
`10,900 (1300)
`
`15 (9)
`
`67.5 (13)
`
`Cl, ml 䡠 kg⫺1 䡠 min⫺1
`ClR, ml䡠 kg⫺1 䡠 min⫺1
`Clm, ml䡠 kg⫺1 䡠 min⫺1
`Frequency ABEC,
`breaths
`Frequency Emax,
`breaths/min
`Frequency Tmax, min
`* Significantly different from GHB alone (P ⬍ 0.05).
`a —, no ABEC, Emax, orT max values could be calculated because respiration is
`similar to the baseline values; SCH50911 completely prevented any significant
`decrease in frequency compared with baseline.
`
`17 (7)
`
`53.0 (19)
`
`—
`
`—
`
`Potential Treatment Strategies. Effects of potential
`treatment strategies on GHB-induced respiratory depression
`are given in Table 4. Administration of 50 mg/kg SCH50911
`5 min after GHB completely reversed the GHB-induced de-
`crease in respiratory rate, as shown in Fig. 4; there was no
`significant decrease in respiratory rate compared with base-
`line after the administration of SCH50911. In fact, a slight,
`but significant, increase in respiratory rate was observed at
`early time points in SCH50911-treated animals. Lower doses
`of 2.5, 5, and 10 mg/kg SCH50911 did not completely reverse
`GHB-induced respiratory depression, and significant de-
`creases in respiratory rate were still observed after antago-
`nist administration. Administration of 10 mg/kg SCH50911
`significantly improved all pharmacodynamic parameters,
`whereas 5 mg/kg improved only the ABEC and Emax and 2.5
`mg/kg had no significant effect on any pharmacodynamic
`parameter compared with that of GHB alone. Administration
`of 50 mg/kg SCH50911 also increased the nonrenal clearance
`
`230
`
`Morse et al.
`
`day precision and accuracy of the quality control samples
`are summarized in Table 1.
`Dose Dependence of GHB Pharmacokinetics/Phar-
`macodynamics. GHB administration in increasing intrave-
`nous doses displayed nonlinear pharmacokinetics, as shown
`in Table 2, similar to previous reports (Lettieri and Fung,
`1979; Morris et al., 2005). Renal clearance and the urinary
`excretion of GHB was almost negligible at the lowest dose of
`200 mg/kg but represented the predominant route of elimi-
`nation at the highest dose of 1500 mg/kg. The pharmacody-
`namic results of this experiment are shown in Fig. 1. Increas-
`ing doses of GHB resulted in a dose-dependent decrease in
`the parameter of respiratory rate, which was accompanied by
`a dose-dependent increase in tidal volume. Minute volume
`was unchanged with the 200 and 600 mg/kg doses but was
`significantly decreased with the 1500 mg/kg dose (95 ⫾ 18
`ml/min at baseline versus Emax of 54 ⫾ 24 ml/min; mean ⫾
`S.D., p ⬍ 0.05). Raw plethysmography traces displaying the
`change in respiratory pattern with GHB administration are
`shown in Fig. 2. As a result of this experiment, respiratory
`rate was considered the primary parameter of interest for
`assessment of receptors involved and potential treatment
`strategies. It was also determined in this experiment that
`1500 mg/kg GHB was the maximal dose that could be admin-
`istered without causing death; therefore, this dose was used
`for further investigation.
`Neurotransmitter Receptors Involved in GHB-In-
`duced Respiratory Depression. Effects of pretreatment
`with specific receptor antagonists are given in Table 3. Ad-
`ministration of the GABAB inhibitor, SCH50911 (150 mg/kg),
`before GHB, resulted in no significant decrease in respiratory
`rate nor a change in tidal volume compared with baseline, as
`displayed in Fig. 3. This inhibitor also increased the nonrenal
`clearance of GHB, but not the total clearance at this dose.
`Administration of the GABAA inhibitor, bicuculline methio-
`dide (5 mg/kg), before GHB, resulted in no change in the
`respiratory effects compared with those for GHB alone and
`had no significant effects on GHB pharmacokinetics.
`
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`Fig. 3. Effect of specific receptor in-
`hibitors on GHB-induced respiratory
`depression. GHB (1500 mg/kg) was ad-
`ministered intravenously, alone and af-
`ter pretreatment with the GABAB re-
`ceptor antagonist SCH50911 (150 mg/
`kg) and the GABAA receptor antagonist
`bicuculline methiodide (5 mg/kg). In-
`hibitors were administered intrave-
`nously 5 min before GHB. Data are pre-
`sented as mean ⫾ S.D.; n ⫽ 3 to 5.
`
`TABLE 4
`Effects of potential treatment strategies on the pharmacokinetics/pharmacodynamics of GHB (1500 mg/kg i.v.)
`Data are presented as mean (SD); n ⫽ 3 to 5. Control ⫽ administration of GHB 1500 mg/kg intravenously. SCH50911 and L-lactate were administered intravenously 5 min
`after GHB. L-Lactate was administered as a 66 mg/kg bolus followed by a 302.5 mg/kg/h infusion for 8 h. One-way analysis of variance followed by Tukey’s post hoc test was
`used to detect statistically significant differences in mean pharmacokinetic and pharmacodynamic parameters.
`
`Control
`
`SCH50911
`
`50 mg/kg
`
`10 mg/kg
`
`5 mg/kg
`
`2.5 mg/kg
`
`L-Lactate
`
`5 mg/kg SCH50911 ⫹
`L-Lactate
`
`Cl, ml 䡠 kg⫺1 䡠 min⫺1
`5.16 (0.70)
`ClR, ml䡠 kg⫺1 䡠 min⫺1
`3.18 (0.66)
`Clm, ml䡠 kg⫺1 䡠 min⫺1
`1.99 (0.17)
`10500 (2700)
`Frequency ABEC, breaths
`Frequency Emax, breaths/min
`17 (7)
`4.35 (1.3)
`Td, h
`a Significantly different from control (P ⬍ 0.05).
`b Significantly different from 5 mg/kg SCH50911 alone (P ⬍ 0.05).
`c Significantly different from L-lactate alone (P ⬍ 0.05).
`d—, no ABEC, Emax, orT d values could be calculated because respiration is similar to the baseline values; no significant decrease in frequency compared with baseline
`was observed after administration of 50 mg/kg SCH50911.
`
`6.17 (0.41)
`3.37 (0.038)
`2.80 (0.52)a
`—d
`—
`—
`
`6.13 (0.22)
`4.03 (0.21)
`2.09 (0.37)
`3690 (1440)a
`51 (3)a
`2.50 (0.20)a
`
`6.13 (0.23)
`3.78 (0.36)
`2.35 (0.17)
`5500 (1440)a
`44 (6)a
`3.15 (0.28)
`
`6.05 (0.69)
`3.56 (0.50)
`2.50 (0.19)
`8720 (513)
`33 (2)
`4.62 (1.2)
`
`6.40 (0.62)a
`4.22 (0.63)a
`2.19 (0.55)
`5470 (1550)a
`24 (5)
`2.45 (0.62)a
`
`7.61 (0.062)a,b,c
`5.28 (0.42)a,b
`2.33 (0.44)
`3170 (957)a
`45 (6)a
`2.17 (0.14)a
`
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`Fig. 4. Effect of potential treatment
`strategies on respiratory rate after
`GHB administration. A, dose-depen-
`dent effects of SCH50911. B, effects of
`L-lactate and L-lactate and SCH50911
`combination therapy. All treatments
`were administered intravenously 5
`min after GHB (1500 mg/kg i.v.). Data
`are presented as mean ⫾ S.D.; n ⫽ 3
`to 5.
`
`of GHB, similar to the higher dose of this receptor antagonist,
`but this effect was not observed at lower doses. Administra-
`tion of the MCT inhibitor L-lactate significantly increased
`GHB renal and total clearance and resulted in significant
`decreases in the frequency ABEC and Td but did not improve
`the Emax. The combined administration of 5 mg/kg SCH50911
`plus L-lactate improved all pharmacodynamic endpoints com-
`pared with those for GHB alone. When L-lactate was admin-
`istered alone at the same dose as that given for GHB, the
`plasma lactate concentrations obtained with this dose were
`much lower in the absence of GHB, indicating an effect of
`GHB on lactate pharmacokinetics. A higher dose of 66 mg/kg
`plus 605 mg/kg/h L-lactate was then administered alone to
`achieve lactate concentrations similar to those obtained with
`GHB administration; ⬃1.5 mM mean increases in plasma
`lactate concentrations were obtained with this higher dose
`and with the lower dose administered concomitantly with
`GHB, as shown in Fig. 5. As displayed in Fig. 6, this higher
`
`dose of L-lactate had no significant effect on respiratory rate,
`tidal volume, or minute volume.
`
`Discussion
`Although abuse of GHB and its precursors has been recog-
`nized, no pharmacologic treatment for GHB overdose exists,
`and current treatment consists only of supportive care. Be-
`cause of the high rates of respiratory depression reported in
`both fatal and nonfatal cases of GHB overdose, this pharma-
`codynamic endpoint serves as a clinically relevant marker for
`the evaluation of potential GHB overdose treatment strate-
`gies. The current data indicate that the primary effect of
`GHB on respiration is a decrease in respiratory rate, accom-
`panied by a compensatory increase in tidal volume, allowing
`minute volume to remain constant until doses approach le-
`thality. This respiratory pattern is similar to that noted in
`some clinical cases of GHB overdose (Mason and Kerns,
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`Fig. 5. Plasma lactate concentrations after administration
`of L-lactate alone and with GHB. Data are presented as
`mean ⫾ S.D.; n ⫽ 4 to 5. L-Lactate low-dose (LD) ⫽ 66
`mg/kg ⫹ 302.5 mg/kg/h. L-Lactate high-dose (HD) ⫽ 66
`mg/kg ⫹ 605 mg/kg/h. L-Lactate LD and HD were admin-
`istered alone at time 0. L-Lactate LD was administered 5
`min after GHB when administered concomitantly.
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`2002), indicating that our rat model is relevant for studying
`this endpoint. When the respiratory rate decreases substan-
`tially and tidal volume reaches a physiologic limit, minute
`volume decreases steeply with increases in GHB concentra-
`tions, resulting in fatality. Although we observed no fatalities
`at doses of 1500 mg/kg i.v. and lower, we observed fatality in
`approximately 50% of animals administered 1750 mg/kg,
`consistent with previous reports indicating that the LD50 of
`GHB administered intravenously in rats is 1700 mg/kg (La-
`borit, 1964). These results indicate that minute volume is an
`insensitive measure of GHB intoxication, because there is
`little change in this measure before death, and the primary
`parameter of interest in GHB-induced respiratory depression
`is respiratory rate.
`Our data indicate that the decrease in respiratory rate
`after GHB administration is mediated primarily by action at
`GABAB receptors, because of complete inhibition of the effect
`of GHB on this parameter with pretreatment of a GABAB
`receptor antagonist, SCH50911. This inhibitor also exhibited
`a surprising effect on GHB pharmacokinetics, significantly
`increasing its nonrenal clearance. This effect was probably
`not translated to increased total clearance because nonrenal
`clearance is a minor route of GHB elimination at 1500 mg/kg
`but may be responsible for some of the effects of this inhibitor
`observed on GHB toxicodynamics in studies at lower GHB
`doses. Although the primary mechanism of GHB-induced
`respiratory depression was evident from GABAB receptor