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Background/Objective: 1,4-BD is a GHB prodrug that produces toxicity after overdose by two GABAergic mechanisms: 1.) GHB interaction with GHB-specific receptors that modulate release of GABA in pathways expressing GABA-B receptors, and 2.) in vivo GHB metabolism by GHB dehydrogenase to GABA. We investigated if the toxicity of 1,4-BD and GHB by GABAergic mechanisms can be decreased with NCS-382 and CGP-35348, novel high affinity antagonists of GHB receptors and GABA-B receptors, respectively. Methods: Male CD-1 mice in group I were pretreated with CGP-35348 200mg/kg i.p. (n = 5) or with control injections of deionized, distilled water (n = 5), followed 10 min. later by 1,4-BD 300mg/kg i.p. ([TD.sub.50] = 170mg/kg for the rotarod test). Mice in group II were pretreated with NCS-382 200mg/kg i.p. (n = 5) or control injections (n = 5), followed 10 min. later by 1,4-BD. Mice in group III received NCS-382 and CGP-35348 (n = 5) or control injections (n = 5), followed 10 min. later by 1,4-BD. Toxicity was then assessed every 10 minutes by the rotarod test (ability of the mouse to log roll on a one in. diameter rod revolving at 6 RPM). Results: CGP-35348 decreased the duration of rotarod failure from 170 minutes in control animals to 80 minutes in pretreated animals. NCS-382 pretreatment did not improve rotarod performance. NCS-382 and CGP-35348 decreased the percentage of animals that failed the rotarod test from 100% in control animals to 40% in pretreated animals. Conclusions: Pretreatment with CGP-35348 and its combination with NCS-382 attenuate 1,4-BD toxicity in CD-1 mice. The potential therapeutic utility of these receptor antagonists in treating patients with 1,4-BD toxicity warrants further examination., Quang L(1), Desai M(2), Boyer E(1), Shannon M(1), Woolf A(1), Maher T(2). (1) Children's Hospital, Massachusetts/Rhode Island Poison Control Center, Harvard Medical School, (2) Massachusetts College of Pharmacy and Health [...]

Background/Objectives: 1,4-BD is abused at dance raves and nightclubs, where ethanol (ETOH) and other drug use is prevalent. We examined interactions between 1,4-BD and ETOH as well as the 'club drugs,' flunitrazepam and ketamine. Methods: Drug doses were derived from our lab (1,4-BD, ETOH) or literature review (flunitrazepam, ketamine) and administered by i.p. route. Male CD-1 mice received 1,4-BD 600mg/kg alone (n = 10), ETOH 2g/kg + 1,4-BD 600mg/kg (n = 10), ETOH 2g/kg + 1,4-BD 1g/kg (n = 10), ketamine 100mg/kg + 1,4-BD 600mg/kg (n = 10), or flunitrazepam 5mg/kg + 1,4-BD 600mg/kg (n = 10). Toxicity was evaluated every 10 min. by the righting reflex and rotarod test. Results: Mice receiving 1,4-BD (600mg/kg) alone, and in combination with flunitrazepam, ketamine, and ETOH, lost their righting reflex for 110, 180, 110, and 0 min., respectively. Mice receiving high dose 1,4-BD (1g/kg) and ETOH lost their righting reflex for 300 min. Mice receiving 1,4-BD (600mg/kg) alone, and in combination with flunitrazepam and ketamine, failed the rotarod test for 210, 240, and 190 min., respectively. Mice receiving 1,4-BD (600mg/kg) and ETOH failed the rotarod test for the initial 50 min., followed by a 50-min. period when mice recovered their ability to perform the rotarod test. However, toxicity then recrudesced with all mice again failing the rotarod test for 120 min. Mice receiving high-dose 1,4-BD (1g/kg) and ETOH failed the rotarod test for a sustained 500 min. Conclusions: For the righting reflex, rank order of toxicity was: 1,4-BD (1g/kg) + ETOH [is greater than] 1,4-BD + flunitrazepam [is greater than] 1,4-BD alone and 1,4-BD + ketamine [is greater than] 1,4-BD (600mg/kg) + ETOH. For the rotarod test, it was: 1,4-BD (1g/kg) + ETOH [is greater than] 1,4-BD + flunitrazepam [is greater than] 1,4-BD alone [is greater than] 1,4-BD + ketamine. 1,4-BD (600mg/ kg) and ETOH recrudescing toxicity may represent dose-dependent, delayed 1,4-BD toxicity from greater alcohol dehydrogenase affinity for ETOH., Quang L(1), Desai M(2), Boyer E(1), Shannon M(1), Woolf A(1), Maher T(2). (1) Children's Hospital, Massachusetts/Rhode Island Poison Control Center, Harvard Medical School, (2) Massachusetts College of Pharmacy and Health [...]

Background/Objectives: GVL, a methylated congener of GBL, now appears on internet 'recipes' as a key ingredient for synthesis of gamma-hydoxyvalerate (GHV), a methylated congener of GHB. GHV has been reported by users to produce GHB-like effects, but GVL effects have not been described. Because GVL is the lactone ring structure of GHV, we hypothesize that it may undergo in vivo biotransformation to GHV by tissue lactonases or nonenzymatic hydrolysis. We investigated the potential of GVL to produce GHB-like toxicity. Methods: Male CD-1 mice received GVL 600mg/kg i.p. (n = 7), 1200mg/kg i.p. (n = 7), 1800mg/kg i.p. (n = 7), or control injections of deionized, distilled water i.p. Toxicity was assessed every 15 min. by observing for seizures, the righting reflex, and rotarod test (ability to log roll for 10 sec. on a 1-in. diameter rod revolving at 6 RPM). Results: Seizures, loss of righting reflex, and failure of the rotarod test were not observed in mice receiving control injections, GVL 600mg/kg, and GVL 1200mg/ kg. Mice receiving GVL 1800mg/kg did not lose their righting reflex, but 6/7 of these mice failed the rotarod test 20 minutes after administration for a total duration of 210 minutes. Conclusions: Previous studies from our lab have observed seizures as well as failure of the righting reflex and rotarod test from the GHB prodrug, 1,4-butanediol (1,4-BD), at doses of 560mg/kg and 170mg/kg, respectively. GVL appears to have less toxicity than 1,4-BD because seizures or loss of the righting reflex were not observed at any dose, and impairment of the rotarod test occurred only at 1800mg/ kg. The methyl group appears to decrease GVL toxicity as a GHB-like prodrug., Quang L(1), Desai M(2), Boyer E(1), Shannon M(1), Woolf A(1), Maher T(2). (1) Children's Hospital, Massachusetts/Rhode Island Poison Control Center, Harvard Medical School, (2) Massachusetts College of Pharmacy and Health [...]

Background/Objective: Published studies have observed 4-MP, an alcohol dehydrogenase (ADH) antagonist, to block toxic manifestations of 1,4-BD in male CD-1 mice. We investigated if these observations corroborate with 1,4-BD and GHB blood concentrations. Methods: Male CD-1 mice were pretreated with 4-MP 25 mg/kg i.p., or deionized, distilled water (controls), followed 5 min. later with 1,4-BD 600mg/kg i.p. (Toxic [Dose.sub.50]). Another group of mice was administered 1,4-BD 600 mg/kg i.p. first, followed 5 min. later with 4-MP 25 mg/kg i.p. as an antidote or deionized, distilled water (controls). After 60 min., all mice were tested for the righting reflex and rotarod test (ability to log roll for 10 sec. on a 1-inch diameter rod revolving at 6 RPM) and sacrificed for blood 1,4-BD and GHB concentrations by GC/ MS. Results: Avg. Avg. BD Level GHB Level Righting Rotarod ([micro]g/mL) ([micro]g/mL) Reflex Test 4-MP Pretreat 1505 9 Present Passed Controls Conclusions: 4-MP, given as a pretreatment and as an antidote, was effective in blocking in vivo biotransformation of 1,4-BD to GHB. The presence of high 1,4-BD blood concentrations did not correlate with failure of the righting reflex or rotarod test. Conversely, the presence of blood GHB concentrations correlated with failure of the righting reflex and rotarod test. Quang L(1), Desai M(2), Kraner J(3), Shannon M(1), Woolf A(1), Maher T(2). (1) Children's Hospital, Massachusetts/Rhode Island Poison Control Center, Harvard Medical School, (2) Massachusetts College of Pharmacy and Health Sciences, Boston, MA, (3) AIT Laboratories, Indianapolis, IN, Quang L(1), Desai M(2), Kraner J(3), Shannon M(1), Woolf A(1), Maher T(2). (1) Children's Hospital, Massachusetts/Rhode Island Poison Control Center, Harvard Medical School, (2) Massachusetts College of Pharmacy and Health [...]

Objective: BD is a prodrug that is biotransformed by alcohol dehydrogenase to gamma-hydroxybutyrate (GHB). This study examined if 4-MP can be used as an antidote for BD toxicity. Methods: Male CD-1 mice were given i.p. injections of BD at 600 mg/kg ([TD.sub.50]) and divided into 3 groups. Group 1 received i.p. 4-MP at 25 mg/kg or deionized, distilled water (controls) 1 minute after BD. Group 2 received the same interventions 5 minutes after BD. Group 3 received the same interventions at the time they failed the roto-rod (RoRo) test after BD. Toxicity was assessed by the righting reflex and roto-rod test at 10-minute intervals up to 3 hours. Results: Conclusion: 4-MP confers an antidotal effect on CD-1 mice for the roto-rod test when given at 1 and 5 minutes postBD, and at onset of BD toxicity, presumably by blocking biotransformation of BD to GHB. Time After 1,4-Butanediol I.P. Injection Group 1 Group 2 Group 3 (1 min) (5 min) (failed RoRo) Righting Reflex 4-MP Group 10/10 10/10 10/10 Control Group 8/10 8/10 7/10 p-value NS NS NS Roto-rod Test 4-MP Group 10/10 10/10 9/10 Control Group 1/10 1/10 1/10 p-value (*) Winner of the American Academy of Clinical Toxicology 1999 Research Award. Quang L, Maher T, Shannon M, Woolf A. Children's Hospital, Massachusetts/Rhode Island Poison Control System, Harvard Medical School, Massachusetts College of Pharmacy and Allied Health Sciences, Boston, MA, (*) Winner of the American Academy of Clinical Toxicology 1999 Research Award. Quang L, Maher T, Shannon M, Woolf A. Children's Hospital, Massachusetts/Rhode Island Poison Control System, Harvard Medical School, [...]

Objective: To determine the Toxic Dose 50 ([TD.sub.50]) of 1,4-butanediol (BD) in CD-1 mice. Methods: Male CD-1 mice received intraperitoneal (ip) injections of BD or deionized, distilled water (BD excipient; controls). Toxicity was observed at 10-minute intervals up to 60 minutes by assessing righting reflex (ability to regain an upright posture within 10 seconds after being placed on its back) and the roto-rod test (ability of the mouse to log roll on a 1-inch diameter rod revolving at 6 RPM). Results: BD Dose (mg/kg) 100 200 300 400 500 Righting Reflex BD Group 8/8 10/10 10/10 10/10 5/10 Control Group 8/8 10/10 10/10 10/10 10/10 p-value NS NS NS NS .01 Roto-rod Test BD Group 8/8 0/10 0/10 0/10 0/10 Control Group 8/8 10/10 10/10 10/10 10/10 p-value NS The [TD.sub.50] of BD for loss of righting reflex is 554 mg/kg (95% CI, 442-693 mg/kg). The [TD.sub.50] of BD for failing the roto-rod test ranges from 100-200mg/kg. The [TD.sub.50] of BD for both loss of the righting reflex and failure of the roto-rod test appears to be 600 mg/kg. The roto-rod test is more sensitive than the righting reflex for detecting BD toxicity. Conclusions: These results are important for animal studies investigating BD toxicity. (*) Winner of the American Academy of Clinical Toxicology 1999 Research Award. Quang L, Maher T, Shannon M, Woolf A. Children's Hospital, Massachusetts/Rhode Island Poison Control System, Harvard Medical School, Massachusetts College of Pharmacy and Allied Health Sciences, Boston, MA, (*) Winner of the American Academy of Clinical Toxicology 1999 Research Award. Quang L, Maher T, Shannon M, Woolf A. Children's Hospital, Massachusetts/Rhode Island Poison Control System, Harvard Medical School, [...]

Background: Among children, iron (Fe)-deficiency anemia and lead (Pb)-poisoning are known to cause cognitive impairment, possibly via effects on dopamine metabolism. The objective of this study was to determine the effects of combined developmental iron deficiency and lead poisoning on spatial learning and to correlate these findings with brain extracellular dopamine (DA) concentrations in rats. Methods: Male rats were exposed prenatally and postnatally with: 1) Fedeficient diet and control water; 2) Fe-deficient diet and 300 ppm Pb in water; 3)Iron replete diet and control water; and 4) Iron replete diet and 300 ppm Pb in water. Morris Water Maze (MWM) testing was performed on the 6th to 7th week of life, followed by DA microdialysis in the nucleus accumbens. Iron deficiency was assessed by hematocrit and blood lead level by Inductively Coupled Plasma-Mass Spectrometry. Multiple linear regression models using MWM probe test results as the dependent variable and hematocrit, blood lead and extracellular DA concentrations as both independent variables and effect modifiers were generated to assess their association with spatial learning. Results: With respect to MWM probe tests, Fe-deficient, and iron deficient/lead poisoned groups performed worse than control diet groups (p [is less than] 0.05 for both). Extracellular DA levels did not differ among the four treatments groups; however, DA concentrations were directly correlated with probe test perlbrmance ([R.sup.2] = 0.24, p [is less than] 0.05). In the multiple regression model, hematocrit, blood lead level and dopamine concentration were all significantly associated with probe test results. No significant interaction terms were generated among the 3 independent variables, suggesting a lack of synergism. Conclusions: Iron deficiency, lead poisoning and extracellular dopamine are all independent predictors of spatial learning. Our results suggest that extracellular dopamine levels do not change with chronic developmental iron deficiency and lead exposure. Chronic lead poisoning and iron deficiency may still influence dopaminergic systems via receptor function or number., Wright RO, Bhole G, Pilipovic L, Amarasiriwardena C, Hu H, Maher T. Department of Pediatrics, Brown Medical School, Providence, RI; Massachusetts College of Pharmacy, Boston, MA; Channing Laboratory, Harvard Medical [...]

Objective: BD is a prodrug that undergoes biotransformation by hepatic alcohol dehydrogenase to gamma-hydroxybuty-rate (GHB). This study investigated if pretreatment of CD-1 mice with 4-MP blocks BD toxicity. Methods: Male CDI mice were pretreated with intraperitoneal (ip) injections of 4-MP (25mg/kg) or deionized, distilled water (controls). Thirty minutes later, mice in both groups were given BD 600-6000 rog/kg ip. Toxicity was assessed by the righting reflex and the roto-rod test at 10-minute intervals up to 60 minutes. Results: Conclusion: Pretreatment of CD-1 mice with 25 mg/kg 4-MP increases the [TD.sub.50] for BD at doses of 600-3000 mg/ kg, presumably by inhibiting alcohol dehydrogenase biotransformation of BD to GHB. (*) Winner of the American Academy of Clinical Toxicology 1999 Research Award. 1,4-BD Dose (mg/kg) 600 700 800 900 Righting Reflex 4-MP Group 10/10 10/10 10/10 10/10 Control Group 2/10 4/10 1/10 2/10 p-value Quang L, Maher T, Shannon M, Woolf A. Children's Hospital, Massachusetts/Rhode Island Poison Control System, Harvard Medical School, Massachusetts College of Pharmacy and Allied Health Sciences, Boston, MA, Quang L, Maher T, Shannon M, Woolf A. Children's Hospital, Massachusetts/Rhode Island Poison Control System, Harvard Medical School, Massachusetts College of Pharmacy and Allied Health Sciences, Boston, [...]

Background: 1,4-BD produces toxicity via GHB interaction at [GABA.sub.B] and GHB receptors. The effects of chronic 1,4-BD abuse are not known. However, chronic administration of [GABA.sub.B]ergic agents have produced learning and memory deficits in rats. Objective: We examined the effect of oral 1,4-BD self-administration by rats on the Morris water maze task. Methods: 24 male SD rats were divided into 4 groups (N = 6 each group). Group 1 (controls) received only tap water in their drinking bottle. Group 2 received 1,4-BD (0.75% w/v) in their drinking bottle. Group 3 was treated with 4-methylpyrazole (4-MP) 25 mg/kg i.p. daily and received 1,4-BD (0.75% w/v) in their drinking bottle. Group 4 was treated with Baclofen 6 mg/kg i.p. daily and received tap water in their drinking bottle. Rats were tested daily for spatial learning and memory by the Morris water maze test for 7 consecutive days. Results: The mean ([+ or -] SEM) daily intake of 1,4-BD in groups 2 and 3 was 1097 [+ or -] 23 mg/kg and 1384 [+ or -] 59 mg/kg, respectively. Latency (seconds) and distance traveled (cm) to reach the submerged platform were significantly increased for groups 2-4 versus controls on day 2. Thereafter, only group 4 continued to exhibit significant deficits. Memory was not impaired in any rat. Conclusion: Oral self-administration of 1,4-BD in drinking water by rats produced an early but transient deficit in spatial learning; 4-MP did not prevent this deficit. Only the pure [GABA.sub.B] agonist, Baclofen, resulted in persistent rat spatial learning impairment. 1,4-BD doses up to 1.4g/kg/d did not produce [GABA.sub.B]ergic-induced learning deficits., Quang L, Desai M, Maher T, Woolf A, Shannon M. Children's Hospital Boston/Harvard Medical School, Massachusetts College of Pharmacy and Health Sciences, Boston, [...]

Background: 1,4-BD and GBL produce toxicity through their common metabolite, GHB, which interacts with GHB and [GABA.sub.B] receptors. Objective: We investigated if 1,4-BD and GBL neurotoxicity can be decreased with SCH-50911, a high affinity selective [GABA.sub.B] receptor antagonist. Methods: For 1,4-BD, 16 male CD-1 mice received 1,4-BD 600 mg/kg i.p. followed 15 minutes later by SCH-50911 30 mg/kg i.p. (N = 8) or control injection (N = 8). For GBL, 16 mice received GBL 750 mg/kg i.p. followed 15 minutes later by SCH-50911 30 mg/kg i.p. (N = 8) or control injection (N = 8). Mice from all groups were then evaluated for neurotoxicity every 15 minutes by the righting reflex (RR), rotarod test (RT), grip strength (GS, peak pull force in lbs.), and open field locomotion (OFL, distance traveled in cm.). Results: 1,4-BD and GBL produced initial deficits for all outcome measures in all mice. SCH-50911 decreased the duration of RR failure for 1,4-BD and GBL from 135 and 180 min., respectively, in controls to 45 min. in treated mice. SCH-50911 decreased the duration of RT failure for 1,4-BD and GBL from 210 and 420 min. in controls to 105 and 90 min. in treated mice, respectively. SCH-50911 promoted more rapid recovery of GS to baseline values versus controls for both 1,4-BD and GBL (P < 0.05 by area-under-the-curve, AUC, analysis). For OFL, SCH-50911 significantly improved the distance traveled by treated mice versus controls (P < 0.05 by AUC analysis) for GBL only. Conclusion: SCH-50911 significantly reverses neurotoxicity related to 1,4-BD and GBL, presumably by antagonizing GHB effects at the [GABA.sub.B] receptor., Quang L, Desai M, Maher T, Woolf A, Shannon M. Children's Hospital Boston/Harvard Medical School, Massachusetts College of Pharmacy and Health Sciences, Boston, [...]

Background: Gamma-valerolactone (GVL) is the methylated congener of the GHB precursor, gamma-butyrolactone (GBL). Like GBL, which is a key ingredient in the illicit manufacture of GHB, GVL appears on internet websites as a key ingredient in the synthesis of methyl-GHB. There are no studies directly comparing toxicity between GBL and GVL. Objective: This study evaluated GBL and GVL dose-response curves for neurotoxicity. Methods: Male CD-1 mice received either GBL or GVL i.p. at graduated doses (25-4800 mg/kg) until all animals were toxic (n = 10 for each group at each dose). For each dose of GBL and GVL, mice were evaluated for neurotoxicity 30 and 60 minutes after injection, respectively, by the righting reflex (RR) and rotarod test (RT). The percent of animals failing the RR and RT were recorded, and full dose-response curves were generated. The T[D.sub.50] of GBL and GVL for the RR and RT were calculated by the Litchfield-Wilcoxon Test and compared by the Z-statistic. Mortality for each group was also recorded for each dose at 24, 48, and 72 hours after administration. Results: The T[D.sub.50] of GBL and GVL for the RR were 366.2 mg/kg (95% CI, 273.6-490.2 mg/kg) and 2799.0 mg/kg (95% CI, 2591.3-3023.3 mg/kg), respectively (P < 0.05). The T[D.sub.50] of GBL and GVL for the RT were 99.0 mg/kg (95% CI, 60.1-163.2 mg/kg) and 2084.8 mg/kg (95% CI, 1948.8-2230.2 mg/kg), respectively (P < 0.05). Only one mouse died from GBL at the dose of 1 g/kg. Conversely, 80-100% of mice died when GVL was administered at doses greater than 2400 mg/kg. Conclusion: GBL has a lower T[D.sub.50] than GVL for the RR and RT, but GVL has a higher mortality rate than GBL., Quang L, Desai M, Maher T, Woolf A, Shannon M. Children's Hospital Boston/Harvard Medical School, Massachusetts College of Pharmacy and Health Sciences, Boston, [...]

Background: 1,4-BD and GBL produce toxicity through their common metabolite, GHB, which interacts with GHB and [GABA.sub.B] receptors. Objective: We investigated if 1,4-BD and GBL neurotoxicity can be decreased with CGP-35348, a selective high affinity post-synaptic [GABA.sub.B] receptor antagonist. Methods: For 1,4-BD, 16 male CD-1 mice received 1,4-BD 600 mg/kg i.p. followed 15 minutes later by CGP-35348 500 mg/kg i.p. (N = 8) or control injection (N = 8). For GBL, 16 mice received GBL 750 mg/kg i.p. followed 15 minutes later by CGP-35348 500 mg/kg i.p. (N = 8) or control injection (N = 8). All mice were then evaluated for neurotoxicity every 15 minutes by the righting reflex (RR), rotarod test (RT), grip strength (GS, peak pull force in lbs.), and open field locomotion (OFL, distance traveled in cm.). Results: 1,4-BD and GBL produced initial deficits for all outcome measures in all mice. CGP-35348 decreased the duration of RR failure for 1,4-BD and GBL from 60 and 180 min. in controls to 45 and 60 min. in treated mice, respectively. CGP-35348 decreased the duration of RT failure for both 1,4-BD and GBL from 180 min. in controls to 60 min. in treated mice. CGP-35348 promoted more rapid recovery of GS to baseline values versus controls for both 1,4-BD and GBL (P < 0.05 by area-under-the-curve, AUC, analysis). For OFL, CGP-35348 significantly improved the distance traveled by treated mice versus controls (P < 0.05 by AUC analysis) for both 1,4-BD and GBL. Conclusion: CGP-35348 significantly reverses neurotoxicity related to 1,4-BD and GBL, presumably by antagonizing GHB effects at the [GABA.sub.B] receptor., Quang L, Desai M, Maher T, Woolf A, Shannon M. Children's Hospital Boston/Harvard Medical School, Massachusetts College of Pharmacy and Health Sciences, Boston, [...]