Interaction of barbiturate analogs with the Torpedo californica nicotinic acetylcholine receptor ion channel.

Arias HR, McCardy EA, Gallagher MJ, Blanton MP.

Department of Pharmacology, School of Medicine, Texas Tech University Health Sciences Center, Lubbock, Texas 79430, USA. phrhra ttuhsc.edu

Barbiturate-induced anesthesia is a complex mechanism that probably involves several ligand-gated ion channel superfamilies. One of these superfamilies includes the archetypical nicotinic acetylcholine receptor (nAChR), in which barbiturates act as noncompetitive antagonists. In this regard, we used the Torpedo californica nAChR and a series of barbiturate analogs to characterize the barbiturate binding site(s) on this superfamily member. [(14)C]Amobarbital binds to one high-affinity (K(d) = 3.7 microM) and several (approximately 11) low-affinity (K(d) = 930 microM) sites on the resting and desensitized nAChRs, respectively. Characteristics of the barbiturate binding site on the resting nAChR include: (1) a tight structure-activity relationship. For example, the barbiturate isobarbital [5-ethyl-5'-(2-methylbutyl) barbituric acid] is >10-fold less potent than its formula isomer amobarbital [5-ethyl-5'-(3-methylbutyl) barbituric acid] in inhibiting [(14)C]amobarbital binding. (2) A binding locus within the pore of the nAChR ion channel. Each of the barbiturate analogs inhibited the binding of [(3)H]tetracaine or photoincorporation of 3-trifluoromethyl-3-(m-[(125)I]iodophenyl) diazirine in a mutually exclusive manner. (3) Stereoselective binding. The R(+)-enantiomers of isobarbital and pentobarbital are approximately 2-fold more potent in inhibiting 3-trifluoromethyl-3-(m-[(125)I]iodophenyl) diazirine photoincorporation than the S(-)-enantiomers. Finally, molecular modeling suggests that within the channel, the pyrimidine ring of the barbiturate is located just above the highly conserved leucine ring (M2--9; e.g., delta Leu-265), whereas the 5' side chain projects downward, and depending upon its conformation, introduces steric hindrance to binding because of the restriction in the lumen of the channel introduced by the leucine side chains.

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Multimodal inclusion complexes between barbiturates and 2-hydroxypropyl-beta-cyclodextrin in aqueous solution: isothermal titration microcalorimetry, (13)C NMR spectrometry, and molecular dynamics simulation.

Aki H, Niiya T, Iwase Y, Yamamoto M.

Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, Fukuoka University, 8-19-1 Nanakuma, Jonan-ku, Fukuoka 814-0180, Japan. akih fukuoka-u.ac.jp

Multiple types (structures) of inclusion complexes between barbiturates and 2-hydroxypropyl-beta-cyclodextrin (HPCD) were evaluated by isothermal titration microcalorimetry and (13)C NMR spectroscopy. The geometries of the inclusion complexes were suggested by molecular dynamics simulation. Barbituric acid (BA), barbital (B), amobarbital (AB), pentobarbital (PB), secobarbital (SB), cyclobarbital (CB), and phenobarbital (PHB) were used as barbiturates with different substituents on the barbituric acid ring and compared for inclusion types in aqueous solution. The association constants (K), stoichiometries, and thermodynamic parameters change in free energy (DeltaG) change in enthalpy (DeltaH), and change in entropy [DeltaS] for each type of complex were determined from the calorimetric data. The inclusion complexation was largely entropy driven because of hydrophobic interactions. The values of K increased in the order BA<B<AB<PB<SB<CB<PHB. Barbiturates, except B and BA, form two types of inclusion complex with a 1:1 stoichiometry in the un-ionized forms. The first type of inclusion complex with high affinity (K(1)) was characterized by small negative values of DeltaH(1) and large positive DeltaS(1), where the substituent R2 of the barbiturate was initially inserted into the cavity of HPCD through hydrophobic interactions. There was a good relationship between DeltaG(1) obtained from the calorimetric data for the first type of inclusion complex and DeltaG(R2) calculated from the changes in (13)C Nuclear Magnetic Resonance (NMR) chemical shifts for the substituent R2 of barbiturates. These types were very stable in aqueous solution at various pHs. The second type of complex, with low affinity (K(2)), was characterized by large negative values of DeltaH(2) and small positive DeltaS(2), reflecting van der Waals' interactions in the un-ionized forms of barbiturates at pH values less than pK(a). The values of K(2) were markedly decreased to <10(3) M(-1) as the barbiturates were ionized over pH 8. Thus, in the second type, the barbituric acid ring contributed to forming the complexes. The geometries were stabilized by hydrogen bond formation between the hetero atoms in the barbituric acid ring and the secondary hydroxyl groups on the rim of the cyclodextrin. The (13)C NMR chemical shifts of C4 and C6 carbons in the barbituric acid ring were moved upfield significantly by the inclusion complexation. On the other hand, B and BA could form only one type of complex, the lid-type supramolecular complex with small association constants. Copyright 2001 Wiley-Liss, Inc.

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Thiopentone and methohexital, but not pentobarbitone, reduce early focal cerebral ischemic injury in rats.

Cole DJ, Cross LM, Drummond JC, Patel PM, Jacobsen WK.

Department of Anesthesiology, Loma Linda University, Loma Linda, California 92354, USA. djcole som.llu.edu

PURPOSE: Although barbiturates are considered to be cerebral protectants, little is known regarding the relative efficacy of different barbiturates to reduce ischemic brain injury. In a model of middle cerebral artery occlusion (MCAo), we compared the relative effects of 1.0 and 0.4 burst-suppression doses of thiopentone, methohexital, and pentobarbitone on cerebral infarct. METHODS: During isoflurane anesthesia, MCAo was achieved via a temporal craniotomy. Thirty minutes before MCAo the rats were randomized to receive one of the following which was maintained throughout the study. Halothane (n=20)-1.2 MAC halothane, thiopentone (n=20), methohexital (n=20), or pentobarbitone (n=20). The first ten animals in each barbiturate group received the respective barbiturate in a dose sufficient to maintain burst-suppression of the electroencephalogram (3-5 bursts x min(-1)). The subsequent ten animals in each barbiturate group received 40% of the burst-suppression dose. After 180 min of MCAo and 120 min of reperfusion, cerebral injury was assessed. RESULTS: For the burst-suppression animals, injury volume (mm3, mean +/- SD) was less in the thiopentone group (88 +/- 14) than the halothane (133 +/- 17), methohexital (126 +/- 19), or pentobarbitone (130 +/- 17) groups (P <0.05). For 0.4 burst-suppression animals, injury volume was less for the methohexital group (70 +/- 22) than the halothane (124 +/- 24), thiopentone (118 +/- 15), or pentobarbitone (121 +/- 20) groups (P <0.05). CONCLUSIONS: These data are inconsistent with the longstanding assumption that electrophysiologically comparable doses of the various classes of barbiturates have equivalent protective efficacy. They in turn suggest that mechanisms other than, or at least in addition to, metabolic suppression may contribute to the protective effect of barbiturates.

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In vitro reaction of barbiturates with formaldehyde.

Gannett PM, Daft JR, James D, Rybeck B, Knopp JB, Tracy TS.

West Virginia University, School of Pharmacy, Basic Pharmaceutical Sciences, Morgantown 26506, USA. pgannett hsc.wvu.edu

Barbiturates are widely used as sedatives, hypnotics, and antiepileptics, and, when coupled with their narrow therapeutic index, the probability that their use will result in accidental or intentional death is significant. When barbiturates are implicated in a murder or suicide, analysis for their presence is often required. Under certain conditions, barbiturates are quite stable, but conditions found in vivo immediately after death or after embalming may promote barbiturate decomposition. If extensive decomposition occurs, analysis for them may be difficult or impossible. Here, the stability of three representative barbiturates, under conditions that model those likely to prevail in vivo shortly after death and after embalming, have been studied. Solutions of phenobarbital were found to slowly decompose in water over the pH range of approximately 3.5 to 9.5. More rapid decomposition occurred at higher pH, and 2-phenylbutyric acid was the main decomposition product. Formaldehyde (5-20%) accelerated the decomposition rate 3-10-fold such that phenobarbital decomposition could be complete after 30 days. In contrast, pentobarbital decomposed roughly 10 times more slowly and secobarbital did not detectably decompose under any of the conditions studied. Thus, certain barbiturates may partially or completely decompose in vivo after death, especially after embalming, and thus analysis for them may lead to false negatives. However, this work shows that analysis for the parent barbiturate or its predicted decomposition product may provide data that will reduce the likelihood of false negatives.

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